Selecting ligand agonists and antagonists

ABSTRACT

We have discovered that growth hormones form ternary complexes with their receptors in which site 1 on the hormone first binds to one molecule of receptor and then hormone site 2 then binds to another molecule of receptor, thereby producing a 1:2 complex. We believe this phenomenon is shared by other ligands having similar conformational structure. Assays based on this phenomenon are useful for identifying ligand agonists and antagonists. Sites 1 and 2 are structurally identified to facilitate generation of amino acid sequence variants of ternary complex-forming ligands. Novel variants of growth hormone, prolactin placental lactogen and other related ligands are provided. As a result of our studies with the ternary complex we have determined that selected antibodies to the receptor for these ligands are capable of acting as ligand agonists or antagonists. Novel growth hormones and novel uses for anti-growth hormone receptor antibodies are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase application ofPCT/US92/03743, filed May 6, 1992, which is a continuation in part ofU.S. application Ser. No. 07/864,120, filed Apr. 6, 1992, now abandoned,which is a continuation in part of U.S. application Ser. No. 07/698,753,filed May 10, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of polypeptide ligand and receptorinteractions. In particular, it relates to the field of selecting andscreening antagonists and agonists for polypeptide ligands.

2. Description of the Background Art

Ligand induced receptor oligomerization has been proposed as a mechanismof signal transduction for the large family of tyrosine kinase receptorsthat contain an extracellular ligand binding domain (for reviews seeYarden, Y., et al., Ann. Rev. Biochem 57:443-478 (1988); Ullrich, A., etal., Cell 61:203-212 [1990]). In these models binding of one hormonemolecule (or subunit) (H) per receptor (R) is thought to induceformation of an H₂ R₂ complex. For example, crosslinking andnon-dissociating electrophoretic studies suggest that epidermal growthfactor (EGF) promotes dimerization of the EGF receptor followed byreceptor autophosphorylation and activation of the intracellulartyrosine kinase (Shector, Y., et al., Nature 278:835-838 (1979);Schreiber, A. B., et al., J. Biol. Chem., 258:846-853 (1983); Yarden,Y., et al., Biochemistry, 26:1434-1442 (1987); Yarden, Y., et al.,Biochemistry 26:1443-1451 (1987). Studies of other tyrosine kinasereceptors including the insulin receptor (Kahn, C. R., et al., Proc.Natl. Acad. Sci. U.S.A. 75:4209-4213 (1978); Kubar J., et al.,Biochemistry 28:1086-1093 (1989): Heffetz, D., et al., J. Biol. Chem.261:889-894 (1986), platelet derived growth factor (PDGF) receptor(Heldin, C. H., et al., J. Biol. Chem. 264:8905-8912 (1989); Hammacher,A., et al., EMBO J. 8:2489-2495 (1989); Seifert, R. A., et al., J.Biol., Chem. 264:8771-8778 (1989)) and insulin-like growth factor(IGF-I) receptor (Ikari, N., et al., Mol. Endocrinol. 2:831-837),indicate that oligomerization of the receptor is tightly coupled to thebiological effect. Other groups have recently crystallized a polypeptidehormone in complex with its extracellular binding domain (Lambert, G.,et al., J. Biol. Chem. 264:12730-12736 (1989); Gunther, N., et al., J.Biol. Chem. 265:22082-22085 (1990)). However, more detailed analyses ofthe structural perturbations and requirements for ligand induced changesin these or other receptors have been hampered because of thecomplexities of these membrane associated systems and the lack ofsuitable quantities of highly purified natural or recombinant receptors.

When purified receptors were available the assay procedures were oftenstructured so that the nature of the hormone-receptor complex was notrecognized. In U.S. Pat. No. 5,057,417, hGH binding assays wereconducted using ¹²⁵ I-hGH competition with cold hGH for binding to theextracellular domain of recombinant hGH receptor (hGHbp), or hGH bindingprotein; the resulting complex was treated with antibody to the hGHbp,plus polyethylene glycol, to precipitate the complex formed. Theseimmunoprecipitation assays suggested that hGH formed a 1:1 complex withhGHbp. This immunoprecipitation assay correctly detected the amount of¹²⁵ I-hGH bound, but it incorrectly indicated a 1:1 molar ratio.

Various solid phase assays for hGH receptor and binding protein havebeen used. Such assays detected the amount of hGH bound but not themolar ratio of hGH to receptor. Binding assays with solid phase or withmembrane fractions containing hGH receptor were not suitable fordetermining the molar ratio of hGH to receptor due to an inability todetect the total amount of active receptor and/or the amount ofendogenous hGH bound. Based upon earlier work, such as with EGF, the artassumed the hGH-receptor complex would be an H₂ R₂ tetramer.

The hGH receptor cloned from human liver (Leung, D. W. et al., Nature,330:537 (1987)) has a single extracellular domain (˜28 kD), atransmembrane segment, and an intracellular domain (˜30 kD) that is nothomologous to any known tyrosine kinase or other protein. Nonetheless,the extracellular portion of the hGH receptor is structurally related tothe extracellular domains of the prolactin receptor (Boutin, J. M. etal., Cell 69:(1988)) and broadly to at least eight other cytokine andrelated receptors. hGHbp expressed in Escherichia coli has been secretedin tens of milligrams per liter (Fuh, G., et al., J. Biol. Chem.,265:3111-3115 (1990)). The highly purified hGHbp retains the samespecificity and high affinity for hGH (K_(D) ˜0.4 nM) as compared to thenatural hGHbp found in serum.

hGH is a member of an homologous hormone family that includes placentallactogens, prolactins, and other genetic and species variants of growthhormone (Nicoll, C. S., et al. Endocrine Reviews, 7:169(1986). hGH isunusual among these in that it exhibits broad species specificity andbinds to either the cloned somatogenic (Leung, D. W. et al. [1987]Nature 330: 537) or prolactin receptor (Boutin, J. M., et al. Cell,53:69). The cloned gene for hGH has been expressed in a secreted form inEschericha coli (Chang, C. N., et al., Gene 55:189 [1987]) and its DNAand amino acid sequence has been reported (Goeddel, et al. Nature,281:544 ([1979); Gray, et al., Gene 39:247[1985]). The three-dimensionalstructure of hGH has not previously been available. However, thethree-dimensional folding pattern for porcine growth hormone (pGH) hasbeen reported at moderate resolution and refinement (Abdel-Meguid, S. S.et al. Proc. Natl. Acad. Sci. U.S.A., 84:6434 [1987]). hGH receptor andantibody binding sites have been identified by homolog-scanningmutagenesis (Cunningham, et al., Science 243:1330, 1989). GH withN-terminal amino acids deleted or varied are known. See Gertler, et al.,Endocrinology 118:720 (1986), Ashkenazi, et al., Endocrinology 121:414(1987), Binder, Mol. Endo.,7:1060-1068 (1990), and WO 90/05185.Antagonist variants of hGH are described by Chen, et al., Mol. Endo.,5(10):1845 (1991) and literature set forth in the bibliography thereof;and WO 91/05853. hGH variants are disclosed by Cunningham, et al.,Science, 244:1081 (1989) and Science 243:1330-1336 (1989).

Since the mode of interaction of many polypeptide ligands with theirreceptors has remained uncertain it has been difficult to engineer aminoacid sequence variants of such ligands to achieve desired properties.Essentially, the art has introduced variation at random, perhaps in somecases with guidance from homology analyses to similarly-acting ligandsor animal analogues, or from analysis of fragments, e.g., trypsin digestfragments. Then the art has screened the candidates for the desiredactivity, e.g., agonist or antagonist activity. The screening methodshave been tedious and expensive, e.g., the use of transgenic animals (WO91/05853). Methods are needed for improving the efficiency of selectionof candidates. In particular, methods are needed for focusing oncandidates likely to be either antagonists or agonists. Antagonists aresubstances that suppress, inhibit or interfere with the biologicalactivity of a native ligand, while agonists exhibit greater activity perse than the native ligand.

It therefore is an object of this invention to provide improved methodsfor the efficient selection of agonist or antagonist polypeptideligands.

It is another object herein to provide a method for detecting ligandsthat form sequential 1:2 complexes with their receptors.

Another object herein is to assay candidate substances for their abilityto interfere with or promote the formation of such 1:2 ligand-receptorcomplexes.

An additional object is to provide amino acid sequence variants ofpolypeptide ligands that are capable of acting as agonists orantagonists.

Other objects, features and characteristics of the present inventionwill become more apparent upon consideration of the followingdescription and the appended claims.

SUMMARY OF THE INVENTION

We have unexpectedly found that growth hormones and the class ofconformational ligands to which they belong are capable of forming 1:2complexes with their receptor in which a first ligand site, site 1,binds to one receptor and then a second ligand site, site 2, binds toanother molecule of receptor, thereby yielding a 1:2 complex. Theligands to which this invention are applicable are monomeric ligandscontaining 4 amphipathic antiparallel alpha-helical domains separatedand terminated at both ends by non-helical amino acid sequences. It isnow possible by analogy to our work with growth hormone, prolactin andplacental lactogen to efficiently design agonist or antagonist aminoacid sequence variants of such ligands by introducing amino acidsequence variation into sites 1 and/or 2 as will be more fully describedbelow.

The two-site complex formation assay is used to screen for substanceswhich are ligand agonists or antagonists. Such substances areessentially unlimited and include organic, non-proteinaceous compoundsas well as amino acid sequence variants of the ligands and bindingprotein or receptor variants.

New amino acid sequence variants of such alpha helical ligands also aredescribed. In particular, antagonists for polypeptide ligands areprovided which comprise an amino acid sequence mutation in site 2 whichreduces or eliminates the affinity of the ligand for receptor at site 2.Ideally, the ligand antagonist analogue will have low or no affinity forreceptor at site 2 and and will have elevated affinity for receptor atsite 1.

Also provided herein are agonist ligand amino acid sequence variantshaving mutations at sites 1 and/or 2 which increase the ligand affinityfor one or both sites. In prefered embodiments, the rate constants forboth sites are selected such that the average residence time of theligand in the dimer complex is greater than or equal to the timerequired for the complex to effect the desired cellular response.Polypeptide agonist variants of the ligand are identified by a methodcomprising (a) introducing a mutation into the ligand to produce anagonist candidate, (b) determining the affinity with which the candidatebinds to the receptor through its first ligand site, (c), determiningthe affinity with which the candidate binds to the receptor through itssecond ligand site, and (d) selecting the candidate as an agonist if itbinds at one or both of the first and second sites with greater affinitythan the native ligand.

In accordance with this invention a method is provided for detecting anagonist or antagonist candidate for a polypeptide ligand, which ligandnormally binds in sequential order first to a receptor polypeptidethrough a first ligand site and secondly to a second copy of thereceptor polypeptide through a second ligand site different from thefirst site, comprising determining the effect of the candidate on theaffinity of the polypeptide ligand for receptor at the ligand's secondreceptor binding site. Site 1 interactions are determined byimmunoprecipitation using a site-2 blocking antibody such as Mab5 asdescribed infra. Alternatively, the amount of wild type ligand thatsubstantially forms only a 1:1 complex with receptor is determined andthen the ability of the candidate to compete with native ligand forreceptor at that proportion is determined. Site 2 interactions areassayed by following the ability of the candidate to form the ternarycomplex.

Where the candidate is a polypeptide analogue of the ligand then onepositively correlates an absence of binding of the analogue at site 2with antagonist activity. The ability to bind with greater affinity thannative ligand to receptor site 2 is correlated with agonist activity.Antagonist and agonist activity are both positively correlated with theability of the candidate to bind at site 1 with greater affinity thannative ligand. Small molecule or other non-analogous candidates areassayed for their ability to promote or suppress binding of nativeligand to sites 1 and/or 2. Antagonists are screened for their abilityto interfere with native ligand-receptor binding at site 2 and/or site1, but preferably site 2. This permits the identification of antagoniststhat do not suppress ligand receptor binding at site 1 but which dointerfere with site 2 binding, using for example as a positive control asite 2 disabled variant of the ligand.

The effect of the candidate can be measured in the presence of thenative polypeptide ligand or in comparison to the activity of the nativepolypeptide ligand. In the first alterative, the effect of the candidateon receptor interactions by the wild type ligand is measured. In thesecond the activity of the wild type ligand is used as a positivecontrol and the receptor binding characteristics of the candidate(usually an amino acid sequence variant of the ligand) are measuredwithout the presence of the wild type ligand. In general, however, theassays for agonist or antagonist candidates are best conducted ascompetition-type assays in the presence of wild type ligand.

We also have determined that selected antibodies capable of binding theGH receptor act as antagonists or agonists of GH. Accordingly, methodsare provided for the antagonism or agonism of GH in the therapy ofgrowth hormone deficiency or excess.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a and 1b. FIG. 1a: Crystals of the complex between hGH and thehGHbp. The hGH/hGHbp complex was prepared by purifying the complex overa Sephadex G75-100 size exclusion column equilibrated in 10 mM Tris (pH8.0) and 100 mM NaCl. The high molecular weight peak containing thecomplex was separated from free hGH, pooled and concentrated. Thecomponents were eluted isocratically (FIG. 1b) with a linearacetonitrile gradient at a flow rate of 1 ml/min. The gradient wasstarted at the arrow and is illustrated with a dashed line; theabsorbance at 214 nm is represented by the solid line.

FIG. 2. Gel filtration chromatography of various ratios of hGHbp and hGHcorresponding to 4:1, 3:1, 2:1, 1:1, 0.5:1. The concentrations of hGH(fixed at 10 μM except at 1:1 and 0.5:1 ratios where hGH was 20 μM and40 μM, respectively) and hGHbp in each mixture were based uponabsorbance at 280 nm. One hundred microliter protein samples wereapplied to a sepharose 12 FPLC column (Pharmacia) and eluted. Peaks weremonitored for absorbance at 280 nm.

FIG. 3. Gel filtration chromatography of a 2:1 ratio of hGHbp:hGH (bold,solid line) and variants of human prolactin or human placental lactogen(other lines) that were engineered to bind to the hGHbp.

FIG. 4. Titration calorimetry of hGH with hGHbp. The hGHbp (at 15 μM in10 mM Tris (pH 8.0)) was placed in a 1.37 ml titration cell (MC2titration calorimeter, Microcal Incorporated, Northampton, Mass.) andequilibrated at 25° C. To this solution hGH (437 μM in 10 mM Tris (pH8.0) was added in 4 μL increments. Each injection occurred over 8seconds with an interval of 5 minutes between each injection.

FIG. 5. Scatchard analysis for binding of hGH to the hGHbp wherecomplexes were precipitated with various anti-hGHbp monoclonalantibodies.

FIG. 6. Circular dichroic spectra in the far UV (FIG. 6A) or near UV(FIG. 6B) of the sum of the individual spectra of hGH and the hGHbpbefore (--) and after ( . . . ) mixing the two at a 1:1 ratio. Far-UVand near-UV spectra were collected at 0.2 nm and 0.5 nm intervals in0.01 cm and 1.0 cm cells, respectively.

FIG. 7. Fluorescence emission spectrum of the sum of the individualspectra of hGH and hGHbp before (--) and after ( . . . ) mixing the twoat a 1:1 ratio.

FIG. 8. Homoquenching of 10 nM S237C-AF by serial addition of hGH. Afterincubation, fluorescence measurements were made at an excitation λ of490 nm and an emission λ of 512 nm (bandwidths are 3 nm and 10 nm,respectively) using a Shimadzu RF5000U Spectrofluorophotometer.

FIG. 9. IC₅₀ determination for hGH induced dimerization of S237C-AF.Serial dilutions (3 fold) of S237C-AF (prepared as described in FIG. 8)in binding buffer (20 mM tris-HCl pH 7.5, 0.1% BSA, 0.02% NaN₃) weremade over a range from 20 nM to 0.08 nM and 1.0 ml aliquots weredispensed to assay tubes. Simularly, hGH was serially diluted, but overa range from 1 μM to 0.004 μM. Aliquots (10 μl) of hGH dilution (giving1:2 molar ratio hGH to S237C-AF) and buffer only were added to theS237C-AF containing assay tubes, mixed and incubated to equilibrium for5 hours at 25° C. in dark. After equilibration, fluorescence wasmeasured as previously described (FIG. 8) except excitation bandwidthwas 10 nM. IC₅₀ values are calculated as the concentration of hGH givinghalf-maximal ΔF/F₀ values as determined from 4 parameter curve fits. AnIC50 of 0.54 (+/-0.14) nM was calculated from the mean of sixindependent experiments.

FIG. 10 Reversal of hGH induced S237C-AF dimerization by excess hGH orhGH mutants. S237C-AF and hGH were diluted in binding buffer to aconcentration of 10 nM and 5 nM, repectively, and 1.0 ml aliquotsdispensed to assay tubes. Serial dilutions of either hGH, mutant, orbuffer only were then added and the mixture incubated to equilibrium for5 hours at 25° C. in the dark, and fluorescence measured as describedfor FIG. 8. Data points are means of triplicate measurements andrepresent: , hGH; , K172A/F176A; , hPL recruit. Error bars give SEM.

FIG. 11 Crystal Structure of hGH(hGHbp)₂. The central top region, inthicker lines, represents the hGH molecule. This hGH molecule is boundto two hGHbp molecules: one at the left hand side, and one at the right.Each of these hGHbp molecules has two domains linked by a single strand;the top domains are at the same height as the hGH molecule, the otherdomains are oriented vertically and stick out towards the bottom of thefigure. These last two domains of the hGHbp contact each other at thevery bottom.

FIG. 12 Weight Gain in Response to Antibody Specific for Growth HormoneReceptor. Monoclonal antibody Mab 263, was administered (1.05 mg/kg) toeight rats, and the excipient alone administered to the control group.Daily weight measurments were taken.

FIG. 13 hGH-induced proliferation of FDC-P1 cells containing thehGH-mG-CSF hybrid receptor (9). Cells were grown in RPMI 1640 mediasupplemented with 10 U/ml IL-3, 10 μM p-mercaptoethanol and 10% fetalbovine serum (FBS) at 37° C., 5% CO₂ (8). Cells were washed with samemedia without IL-3 and seeded in 96-well plates in 100 μl aliquots at adensity of 4×10⁵ /ml (◯), 2×10⁵ /ml ( ), and 1×10⁵ /ml (□) prior totreatment with increasing concentrations of hGH for 18 h. To measure DNAsynthesis cells were pulsed with [³ H]-thymidine by addition of 1μCi/well in 20 μl media. After 4 h, cells were harvested and washed onglass filters. Two milliliters of scintillation cocktail were added andcounted with Beckman LS1701 scintillation counter. Each data pointrepresents the mean of triplicate determinations and error bars are theS.D.

FIG. 14 Antagonism of hGH-induced cell proliferation by hGH variants.Cells were prepared as in FIG. 13 and incubated with 1 nM hGH plusincreasing concentrations of the Site 1 mutant (K172A/F176A) ( ), theSite 2 mutant (G120R) (□), the combined enhanced Site 1 mutant/Site 2mutant (H21A/R64K/E174A/G120R)( ), and wild-type hGH (◯).

FIGS. 15a, 15b and 15c depict wheel plots of postulated α helical sitessuitable for variation in preparing IL-6 antagonists or agonists. Notethat residue numbering for these figures commences with the N-terminalpre residue.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The method of this invention facilitates the identification of agonistsor antagonists polypeptide ligands. In general, the method is practicedas follows.

First, one determines the stoichiometry of association of the ligandwith its receptor in order to identify ligands that enter into 1:2ternary complexes with their receptor. The stoichiometry of associationis determined by measuring the proportion of ligand to receptor underphysiological conditions using one of the methods set forth below, e.g.,X-ray crystallography, size exclusion chromatography on Separose gels,antibody binding studies, scanning calorimetry, BIA-core analysis, andCD or fluorescence spectral analysis. It should be noted that an X-raycrystal structure of the ligand-receptor complex is useful but by nomeans required in order to determine the stoichiometry of description.Preferably, the fluorescein homoquenching method described furtherherein is used. In this method, the receptor is labelled withfluorescein positioned such that when two molecules of receptor arebound by ligand as determined by titration the fluorescein moleculesquench one another and the fluorescence of the solution is decreases.The analytical techniques described herein are well known per se andwithin the skill of those in the art.

Preferably, the extracellular domain of the receptor is used in thestoichiometry analysis, i.e., the analysis is conducted in solution invitro with receptor variants having their transmembrane domain deletedor otherwise rendered incapable of membrane insertion or hydrophobicassociation. Optionally, the cytoplasmic region also is deleted. Suchreceptors are known and can be expressed in and recovered fromrecombinant cell culture.

If the receptor contains more than one polypeptide chain then preferablythe receptor preparation contains all of the receptor polypeptidechains. Also, if the ternary complex consists of two different receptorchains (as for example in the case of the IL-2 or IL-3 receptor complexcontaining alpha and beta chains, e.g., Teshigawa, et al., J. Exp. Med.,165:223-238 [1978]) then the analysis is conducted with both chains. Inthis instance the binding to each receptor chain proceeds sequentiallyin one order only. The order of association is readily determined whendifferent receptor molecules are involved.

Some receptors may form a 1:2 complex, but the receptors may eachcontain more than one chain. The use of multichain receptors may not benecessary if the ligand only binds to one of the chains and the other isnot required for the maintenance of proper conformation of the bindingchain; only the chains necessary for ligand binding need to be present.

Ligands forming 1:2 complexes bind to their receptors through twodiscrete binding sites. It is a significant feature of this inventionthat the receptors have been found to bind to these two sites insequential order, first one site (site 1) and then the other (site 2).The reverse order has not been found to occur. This understanding isespecially important for the preparation of antagonist ligands. It isimportant to preserve, if not enhance, the affinity of the ligand forthe first site. Otherwise, the ligand analogue never binds receptor atall. On the other hand effective destruction or inhibition of the secondsite binding is predicate for antagonist activity.

In accordance with this invention the ligand binding sites and theirorder of addition are determined. Sites 1 and 2 are identified bycomparing the conformation of the candidate ligand with that of growthhormone in the fashion more fully described below. They are more fullyresolved by alanine scanning or other systematic mutagensis method,e.g., cassette mutagenesis, or PCR mutagenesis. Conformational analysisallows the field of potential sites 1 and 2 residues to be narrowed downconsiderably before making and screening variants.

In either case, a ligand analogue with a disabling site 2 mutation butfunctional site 1 is identified by its inability to form the ternaryligand:receptor complex although such a variant will be capable offorming a 1:1 complex with receptor. On the other hand an analogue witha disabling site 1 mutation but functional site 2 will be unable to bindto receptor at all. The assay employed for this determination is anyassay that will detect association of polypeptides; the homoquenchingassay described infra is acceptable, as is gel filtration and the like.

Conformation analysis facilitated the selection of ligand candidatesfrom the class of amphipathic alpha-helical monomeric ligands. Sincesuch ligands are conformationally related to growth hormones, placentallactogen and prolactin it is straight-forward to determine sites 1 and 2for these ligands by analogy to the growth hormone structure.Ironically, the primary amino acid sequences of such ligands as EPO,alpha interferon, beta interferon, GM-CSF, G-CSF, and interleukins 2, 3,4, 6 and 7 are poorly homologous to growth hormones, placental lactogenor prolactin. However, when these ligands (which generally are cytokinesor hormones) are analyzed by conventional conformational structurepriniciples (Bazan, et al., Immunol. Today, 11:350-354 (1991) Chou, etal., Biochemistry, 13:222[1974]); they are shown to exhibit certaincommon structural features. Most notably, they are characterized by 4dominant amphipathic alpha helices, each preceded and followed bysubstantially non-helical structure (loops between helices and N- andC-terminal sequence at the protein terminii). The dominant alpha helicestypically are about 15-30 residues in length. They are designated A-D,designated in order from the N-terminus. Short helical segments may bepresent in the loops joining dominant helices.

The alpha-helices of this class of ligands are amphipathic, i.e., theygenerally contain hydrophobic residues on one side of the helix andhydrophilic residues on the opposite side of the helix. Each 3.6successive residues of the helix is termed a turn, in the sense of aspiral ladder. Some minor fraying in the terminii of the helices is tobe expected, i.e., each alpha helical terminus may be varied by about1-3 residues depending upon the algorithm employed and the discretion ofthe artisan. Despite the overall lack of homology among this group ofligands some conserved residues may be found in the helices and thesecan be used to assist in the structural alignment of the ligands withgrowth hormone.

Our analysis of growth hormone and the homologous ligands prolactin andplacental lactogen demonstrated that site 2 for this group ofquaternary-alpha helical cyokines and hormones principally is comprisedby (a) the sequence extending from the N-terminus to about the first 3-4turns of helix A and (b) about the middle 4-5 turns of helix C. Thus,site 2 is discontinuous but both segments are in close proximity in theprotein and in that fashion interact with the receptor. Either or bothsite 2 domains are mutated. The helical hydrophobic residues generallyare ignored for purposes of selecting candidate residues formutagenesis, although occasionally they may affect the functionalintegrity of the candidate. In addition, not all residues within site 2will exhibit functional or structural involvement in receptor binding.However, application of the principles herein greatly reduces the numberof candidates that need to be screened for antagonist or agonistactivity.

Antagonist variants are characterized by substantial changes in thecharge, hydrophobicity or bulk of the native residue at the candidatelocation, as is more fully described below. This generally isaccomplished by substituting the residue in question or by deleting it.In some instances the desired effect can be achieved by inserting aresidue adjacent to a functionally or structurally active site 2residue. The object in the preparation of antagonists is to eliminatereceptor binding at site 2, or to reduce it at least about 2 fold inrelation to native ligand. This is most effectively accomplished byradically changing the character of one or more of the native residuesthat are important in structural interactions (hydrogen bonding, saltbridging, hydrophobic interactions and the like) with the receptor. Suchresidues are called structural residues. Alteratively, a residue isselected at a location, that does not directly contact receptor, butwhich is important in the proper positioning of a residue that doesparticipate in a contact interaction. Such residues are termedfunctional residues.

Typically, no more than about 20 locations (residues) will be ofpotential interest in generating site 2 variants (excluding hydrophobicamphipathic residues). Of these, only a representative member of eachamino acid group is employed in creating candidates, i.e., it is notordinarily necessary to screen 19 variants, representing the remaining19 naturally occuring residues, for each residue within site 2. Instead,representative members of residue groups are selected. Generally, thesegroups are (a) positively charged residues (K, R and H), (b) negativelycharged residues (D and E), (c) amides (N and Q), (d) aromatics (F, Yand W), (e) hydrophobics (P, G, A, V, L, I and M) and (f) unchargedhydrophilic residues (S and T). Further, when preparing antagonistcandidates, rather than screening 5 class-representative residuestypically it is satisfactory to select only 1-3 classes because anysubstantial variation at the appropriate residue(s) will disable site 2.See Table 1a below. The most extreme substitutions are produced byselecting opposed combinations of features, e.g., if the native residueis alanine (small hydrophobic), then an extreme substituent would beglutamic acid, which is hydrophilic, bulky and charged. Further,residues are selected from those which show evolutionary diversity.i.e., if an animal species ligand fails to bind to human receptor site 2then variant residues are selected as candidates. Thus, an adequate poolof mutants likely to contain an antagonist typically will contain aboutfrom 20 to about 60 site 2 variants. A slightly different strategy isused to select candidates for agonists at site 2. See the discussionbelow with respect to site 1. Producing and screening such pools wouldnot involve undue experimentation and would be well within the ordinaryskill in the art.

The selection of an amino acid for substitution also should take intoaccount whether the residue is located within an alpha helix or anonhelical structure. If the residue is part of a helical turn then thesubstituent preferably is not a helix breaker such as proline orglycine. On the other hand, if proline or glycine are the residues foundin a wild type helix then they may be freely substituted since theirsubstitution will not destabilize the helical conformation.

Site 1 also is a discontinuous site. It consists of three segmentslocated (a) in the middle 40% of helix A (perhaps overlapping with theC-terminus of site 2 in helix A), (b) the C-terminal 2/3rds (preferablyC-terminal 1/2) of the loop linking helices A and B, and (c) theC-terminal 1/2 (preferably 1/3) of helix D. The proportions refer to thelinear sequence of amino acid residues. In contrast to the strategy tobe used with site 2 antagonist mutations, the residues falling withinthe site 1 domains remain unmodified (in the case of antagonists, inwhich only site 2 is disabled by mutation) or, if modified, the changesto site 1 are selected so as to not disrupt binding. The reason is thatit is not desirable in most embodiments to disable site 1. Instead, theobjective is to increase site 1 affinity by about 10% to greater than 2fold. Thus, residues within these domains generally are substituted(rather than deleted or subject to adjacent insertion), and the initialscreen is with an alanine scan in order to identify hindrancedeterminants (residues whose bulky side chains, particularly whencharged, hinder or inhibit the ligand-receptor binding interactions).Once hindrance residues are identified, site 1 substitutions for eitheragonists or antagonists are selected from Table 1 a under the heading"agonist" substitutions. Species diversity analysis also will be helpfulin identifying agonists as well. Again, no more than about 20 locationswill need to be selected for site 1 variation. Generally at eachlocation the mutation will be substitution with the remaining members ofthe original residue's group and the residues of the next most closelyrelated group (Table 1a), which contain less bulky side chains and/orare unchanged.

                  TABLE 1a                                                        ______________________________________                                        Table of Candidate Substitutions                                              Site 2                                                                               Agonist       Antagonist                                                        Exemplary           Exemplary                                                                             Preferred                                Wild Type                                                                              Group*    Preferred Group*  Group*                                   ______________________________________                                        Ala (A)  e,f       S         a,b,c,d,                                                                              d                                        Arg (R)  a         K,S,A     b,d,e   b                                        Asn (N)  a,c,      Q,S,A     b,d,e   b                                        Asp (D)  b,c       E,S,A     a,d,e   a                                        Cys (C)  f,e       A,S       a,b,c,d d                                        Gln (Q)  a,c       N,S,A     b,d,e   b                                        Glu (E)  b,c       D,S,A     a,d,e   a                                        Gly (G)  e,f       P,A       a,b,c,d d                                        His (H)  a         E,R,S,A   b,d,e   b                                        Ile (I)  e         L,I,V,A   a,b,c,d,f                                                                             a,b,c                                    Leu (L)  e         I,L,V,A   a,b,c,d,f                                                                             a,b,c                                    lys (K)  a         R,S,A     b,d,e   b                                        Met (M)  e         L,I,V,A   a,b,c,d,f                                                                             a,b,c                                    Phe (F)  a,d       I,L,Y,V,A b,c,e,f f                                        Pro (P)  a,d       G,F,A     b,c,e,f f                                        Ser (S)  a,f       A,T       a,b,c,d,e                                                                             d                                        Thr (T)  a,f       S,A       a,b,c,d,e                                                                             d                                        Trp (W)  d         F,A       a,b,c,e,f                                                                             a                                        Tyr (Y)  d         L,I,F,A,V a,b,c,e,f                                                                             a                                        Val (V)  e         L,I,A,S   a,b,c,d,f                                                                             a,b,c                                    ______________________________________                                         *enumerated groups exclude designated wildtype residue; members of groups     are listed above in the text.                                            

Since each site contains several discontinuous domains variation isintroduced into any one of the domains, i.e., it is not necessary tovary each domain of a given site. The helical domains of site 2 (helix Aor C), preferably helix C, are the preferred mutagenesis locations forsite 2. The helical domains of site 1 (helix A or D), preferably helixD, are the preferred locations for variation in site 1. Typically, only1 residue is varied for each site, although it is within the scope ofthis invention to vary at least 1 residue in each domain of each site (2for site 2, 3 for site 1). In other embodiments. 2 or more residues,usually up to about 5 residues, are varied at each domain.

Helical residue selection for mutagenesis or variation is facilitated byconstruction of helical wheel diagrams such as are shown in FIGS. 15a, band c. These are prepared in conventional fashion and are useful inidentifying target locations for variation in the helical portions ofsites 1 and 2. Particular residues of interest are hydrophilic residues,non-bulky residues or residues that tend to destabilize the helicalconformation.

While substitutions, insertions, deletions or combinations thereof areuseful in preparing candidates for screening, the effect of the residuechanges may extend beyond the residue changes per se. For example, asuitable method for modifying site 2 to prevent receptor binding is tointroduce an N- or O-linked glycosylation site within site 2. The sitewill be glycosylated when expressed in yeast or higher eukaryotic cells,and will interfere with site 2 binding by steric hindrance. Oneadvantage of this approach is that it is not necessary to determine theexact location of site 2 structural residues since insertion of aneighboring bulky group may be all that is required to inhibit binding.Other advantages are the ability to modulate, e.g., increase, thecirculating half life and to reduce immunogenicity of the variant. Thus,for example, the invention includes the insertion of a glycosylationsite in helices A and/or C (preferably C) of the ligands herein. e.g.,IL-2.

The stable of candidate agonists or antagonists then is screened for theability of the candidates to functionally act as agonists orantagonists. Such assays are conventional and widely available e.g.,conventional assays typically used to assay potency and activity of thewild type ligand. Alternatively or in addition, the assays employed todetermine receptor stoichiometry can be used (particularly to identifyantagonists which bind at site 1 but not site 2). These assays per seare routine and do not require undue experimentation.

With respect to human growth hormone, site 2 contact residues includeT3, I4, L6, L9, N12, L15, R16, R19, Q22, Y103, N109, D116, E119, G120,and T123. Site 2 functional residues include F1, I4, L6, R8, D116 andE119. Any residue is substituted at any one or more of these locations,the native residue is deleted or another residue is inserted adjacentthereto. As noted above, members of the same or different class aresubstituted, depending upon whether an antagonist or agonist affect issought. Variation introduced into or adjacent to one or more of theselocations will affect site 2 binding. In general, prefered residues formutation include at least one mutation in the designated regions in theN-terminal domain/helix A and another in the C helix, especially F1, I4,L6, D119 and G120. Examples of hGH antagonists include I 4A/L6A/G120AhGH, I4A/L6A/G120A/T123A hGH, F1A/I4A/G120I/T123A hGH, F1A/I4A/G120FhGH, and F1T/I4F/L6R/G120R/T123D hGH, as well as any of the foregoingwith an additional mutation at a residue such as E 174, H21, R64, K172,and/or F176 that increases the affinity of site 1 for its receptor. Forexample, E174 preferably is mutated to S, but also is mutated in otherembodiments to a residue selected from G, V, L, I, A, T, D, N, Q, H, K,R, M, F, Y, W or P. F176 preferably is mutated to Y, and is optimallyused in combination with E174S, R168N, D171S/A and/or I179T (from helixD) and, from helix A, F10A, M14W, H18D and H21N. Two site 1 hGH variantshave been identified by phagemid screening that exhibit about 30 timestighter binding for the GHbp that does the wild type hormone:F10A/M14W/H18D/H21N/R167N/D171S or A/E174S/F176Y/I179T. Mutations atthese sites are combined with the above-noted mutations at site 2 inorder to produce agonists or antagonists. Examples of antagonistsinclude F1A/I4A/F10A/M14W/H18D/H21N/G12OR, F, Y, W, D, E orI/R167N/D171S or A/E174S or A/F176Y/I179T hGH; F1A/I4A/H21A/R64K/E174AhGH, I4A/G120R/E174A hGH and I4A/G120I/E174A hGH.

In other antagonist embodiments, one or more residues selected from thegroup consisting of I4A, L6A, F1, and G120 are deleted while theremaining residues are substituted, and/or one or more residues areinserted adjacent to these residues. Combinations of substitutions,deletions and insertions are useful. Selecting them simply will be amatter of optimizing the activity of the growth hormone. Examples ofsuch combinations include F1(Δ)/I4A/G120I/E174A, andI4(Δ)/G120(K)/E174A.

The effect of mutations at locations in sites 1 and 2 generally will beto depress binding and affinity, although selected modifications atthese sites alternatively may lead to increases in affinity asdetermined by routine screening. For example, variation at E174 (S, G. Aor T) and at positions 21, 18 and 64 have been shown to increase site 1affinity for GHbp.

HGH site 1 contact residues are H18, H21, Q22, F25, K41, Y42, L45, Q46,P61, S62, N63, R64, E66, R167, K168, D171, K172, T175, R178, and C189.Residues having side chains that affect the function of site 1 are P5,L6, F10, M14, F54, E56, I58, S62, N63, R64, E66, Q68, Y164, D171, K172,E174, T175, F176, R178, I179, C182 and V185. Preferred residues forincreasing the affinity of site 1 for receptor are H21, R64 and E174. Ingeneral, site 1 residues are only substituted, and not deleted, nor areresidues inserted adjacent thereto. Further, the site 1 substitutionsgenerally will be drawn from the same group as the native residue or aclosely related group, as noted in Table 1; ordinarily it is notdesirable to heavily perturb site 1 since both agonist and antagonistactivity require that site 1 bind to receptor. Nonetheless, exceptionsdo exist, for example E174A, so it is desirable to screen a panel ofsubstitutions to determine the optimal one.

Analogous residues in other growth hormones are easily identified andmodified in the same fashion. For example, I4 in hGH corresponds to M4in bGH. Note that some variation in residue numbers may exist incomparing growth hormones from other species as well as other alleles ofhGH. If the animal GH does not contain the same residue as human GH atthe homologous position then the substituted residue is one that isdifferent from the animal residue and preferably different from humanresidue at that location. Otherwise, the selection of residues formutagensis is conducted in the same fashion as described above.

Structural analysis or molecular modeling is used to identify analogoussequences for variation in other ligands, i.e., monomeric polypeptideligands containing 4 antiparallel amphipathic alpha helices. Thestructure of the candidate ligand is determined using Chow Fassmananalysis and then analogous residues located within sites 1 and 2 foreach ligand are identified. In some instances, structural studies havealready been published, and all that is needed is to compare theresidues in the various domains with growth hormones in order toidentify sites 1 and 2. Monomeric ligands are those which are found asmonomers in circulation under normal physiological conditions.

Presently known examples of such ligands include EPO, GM-CSF, G-CSF,interleukins 2, 3, 4, 6 and 7, placental lactogen and prolactin,alpha-interferon, beta interferon. Others may be identified in thefuture and the teachings of this invention are equally applicablethereto.

In order to produce antagonist candidates for these ligands, substantialmutations are introduced into one or both of two regions: (a) from theN-terminus to the first N-terminal 1/3 of the A helix and (b) about themiddle 1/2 (preferably 1/3) of the C helix. These domains correspond tothe site 2 domain of hGH. Agonists are made by introducing less bulkyand/or less charged substitutions into sites 1A and/or 2 (See Table 1).Optimal antagonists are produced by mutating site 2 to prevent orsubstantially delete receptor binding and by mutating site 1 to increaseits affinity for receptor. As can be seen, site 1 is modified toincrease its affinity for receptor in both the agonist and antagonistembodiment.

As an illustration of the manner in which antagonists and agonistvariants of ligands other than growth hormone are prepared, reference ismade to Table 1b below which discloses the postulated site 1 and 2principal and core determinants for hPRL, IL-2, IL-3, IL-4, IL-6,GM-CSF. G-CSF and EPO. The helical determinants for each site wereselected by identifying the helical domains of each ligand and comparingthem with the analogous domains of hGH. The same analysis is applied inidentifying non-helical domains that contribute to the structure orfunction of ligands other than hGH. Table 1b reflects our belief thatantagonist variants preferably are made by targeting site 2 helicalresidues rather than site 2 domains in the N-terminii. However,non-helical analogous residues for sites 1 and 2 may also varied. TheTable 1b residues postulated for sites 1 and 2 are believed to containat least one residue that structurally or functionally interacts withthe receptors for the tabulated ligands. This is readily confirmed byblock alanine scanning or homolog-scanning of 1 to 3 of the 3 helicaldomains or portions thereof, deleting the domains or portions thereof,or substituting residues within one or more of the 3 domains so as tocreate an O- or N-linked glycosylation site. Once the activity of thedomain is confirmed, it is a straight-forward matter to useresidue-by-residue alanine-scanning to identify the key functional orstructural residues.

                                      TABLE 1b                                    __________________________________________________________________________    Locations of helical segments and approximate sites 1 and 2 determinants      in helices of                                                                 various ligands.                                                              Hormone                                                                            Helix 1                                                                           length                                                                            Site 2                                                                             Helix 2                                                                            Helix 3                                                                            Length                                                                            Site 2                                                                             Helix 4                                                                            Length                                                                            Site 1                          __________________________________________________________________________    hGH   6-33*                                                                            (28)                                                                               8-19                                                                              72-92                                                                              106-128                                                                            (23)                                                                              116-123                                                                            155-184                                                                            (30)                                                                              164-183                         hPRL 15-42                                                                             (29)                                                                              17-28                                                                               79-104                                                                            114-138                                                                            (25)                                                                              125-132                                                                            162-193                                                                            (32)                                                                              172-192                         IL-2 12-27                                                                             (18)                                                                              .sup. 13-20.sup.+                                                                  52-73                                                                              84-97                                                                              (14)                                                                              .sup. 90-95.sup.+                                                                  115-133                                                                            (19)                                                                              121-133                         IL-3 18-27                                                                             (10)                                                                              19-23                                                                              56-66                                                                              69-81                                                                              (13)                                                                              75-79                                                                              105-120                                                                            (16)                                                                              110-119                         IL-4  5-17                                                                             (13)                                                                               6-11                                                                              40-56                                                                              72-90                                                                              (19)                                                                              81-87                                                                              110-125                                                                            (16)                                                                              115-124                         IL-6 18-43                                                                             (26)                                                                              20-30                                                                               80-102                                                                            110-134                                                                            (25)                                                                              121-130                                                                            154-184                                                                            (31)                                                                              163-183                         GM-CSF                                                                             13-28                                                                             (16)                                                                              14-21                                                                              55-64                                                                              74-87                                                                              (14)                                                                              80-85                                                                              103- 116                                                                           (14)                                                                              108-115                         G-CSF                                                                              10-35                                                                             (26)                                                                              12-22                                                                              73-96                                                                              105-127                                                                            (23)                                                                              116-123                                                                            148-176                                                                            (29)                                                                              157-175                         EPO   4-28                                                                             (25)                                                                               6-16                                                                              58-82                                                                               89-113                                                                            (25)                                                                              101-109                                                                            136-160                                                                            (27)                                                                              145-159                         __________________________________________________________________________     *Residues numbered from first Nterminal mature residue                        .sup.+ Site 2 for IL2 is believed to bind to the IL2 receptor beta chain.     Antagonists are conveniently assayed by forming a 1:1 complex of IL2          candidate with IL2 receptor alpha chain and screening for the ability of      the 1:1 complex to bind beta chain. Similarly. site 1 agonists are            identified by screening for their ability to compete with native IL2 for      the receptor alpha chain.                                                

Helix structural information for Table 1b was obtained from DeVos, etal., Science, 225:306-312 (1992)(hGH); Bazan, et al., Immunol. Today,11:350-354 (1991)(hPRL, IL-6, IL-2 and EPO): Lokker, et al., EMBO J.,10:2125-2131 (1991); Bazan, et al., op cit. (IL-2); and Diederichs, etal., Science 254:1779-1782 (GM-CSF). As noted above, such informationfor other ligands if obtained by modeling, nmr, or preferably, by x-raycrystallographic analysis.

The site designations in Table 1b were arrived at by calculating contactpatches based on site 2 being 0.07 to 0.5 of the length of helix A and0.5 to 0.8 of the length of helix C. These sites should be consideredapproximate and likely will require modest refinement; each site may bepositioned ±1-5 residues from the sequence noted.

In the preparation of antagonists the residues or structures introducedinto the target helical residues generally are not of the same class(see supra) and preferably are more bulky than the residue for whichthey are substituted. In other embodiments, the site residues aredeleted, or other residues (such as helix breakers like G or P) areinserted adjacent to a site residue. A group of candidates then areprepared and screened by the methods set forth herein to identifyoptimal candidates. It should be emphasized, however, that even if avariant ligand fails to act as an agonist or antagonist in comparisonwith native ligand, the variant is useful for the conventional uses forthe ligand (where the variant retains approximately the same activity asthe native ligand) or as an immunological, e.g., diagnostic, reagentwhere it is unable to bind to receptor.

A representative scheme for preparation of IL-2 antagonist candidates isshown in Table Ic. In this scheme, putative site 2 residues asidentified in Table Ib are substituted with preferred amino acids forreduced receptor binding. Addtional alternative substitutions are alsoindicated in the Table.

                                      TABLE Ic                                    __________________________________________________________________________    Substitutional Mutations for Production of IL-2 Antagonist Candidates                    Preferred                                                          Residue                                                                            Wild-type                                                                           Substitutions                                                                        Alternative Substitutions                                   __________________________________________________________________________    13   Q     A,R    N,D,B,C,E,Z,G,H,I,L,M,F,P,S,T,W,Y,V                         14   L     A      R,N,D,B,C,Q,E,Z,G,H,I,K,M,F,P,S,W,Y,V                       15   E     A,R    N,D,B,C,Q,Z,G,H,I,L,M,F,P,S,T,W,Y,V                         16   H     A,R    N,B,C,Q,E,Z,G,I,L,K,M,F,P,S,T,W,Y,V                         17   L     A,R    D,B,C,Q,E,Z,G,H,I,K,M,F,P,S,T,W,Y                           18   L     A      R,N,D,B,C,Q,E,Z,G,H,I,K,M,F,P,S,W,Y,V                       19   L     A      R,N,D,B,C,Q,E,Z,G,H,I,K,M,F,P,S,W,Y,V                       20   D     A,R    N,B,C,Q,Z,G,H,I,L,K,M,F,P,S,T,W,Y,V                         90   N     A,R    D,B,C,Q,E,Z,G,H,I,L,K,M,F,P,S,T,W,Y,V                       91   V     A,W    R,N,D,B,C,Q,E,Z,G,H,I,L,K,M,F,P,S,T,Y                       92   I     A,W    R,N,D,B,C,Q,E,Z,G,H,L,K,M,F,P,S,T,Y,V                       93   V     A,W    R,N,D,B,C,Q,E,Z,G,H,I,L,K,M,F,P,S,T,Y                       94   L     A,W    R,N,D,B,C,Q,E,Z,G,H,I,K,M,F,P,S,T,Y,V                       95   E     A,R    N,D,B,C,Q,Z,G,H,I,L,K,M,F,P,S,T,W,Y,V                       __________________________________________________________________________

Representative helical wheel plots for IL-6 helices A, C and D aredepicted in FIGS. 5a, b and c, respectively. Of potential interest inhelix A are D54, R58, E51, K55, T48, R52, Q56 and S49, and in helix Care K157, A158, Q155, Q152, K156 and V149.

A similar plot for hPRL suggests that locations for modifying site 2 areresidues H58, D69, H55, D48, N59, V52, D45, Y56 and R49 (helix A) andK143, Q150, G157, E146, Q164, R153, L160, S142, E149, S163, V145, K152,E148, H166, E156 and E159 (helix C).

hPRL site 1 (helix D) residues of interest are C202, R220, S191, K209,N198, L216, R205, S194, N212, H201, C219, E190, H208, Y197, K215, R204,D211, L193, L200 and K218. hPRL residues are numbered with theN-terminal M of the presequence=1.

Of course, the agonists and antagonists herein also include ligands inwhich variation is introduced into additional residues than those foundin sites 1 and/or 2. For example, the candidates are fused to otherpolypeptides, e.g., to facilitate immunoaffinity purification, haveregions or residues deleted, substituted or inserted which do notparticipate in the desired activity and are not required for maintenanceof the proper conformation, e.g., to make active fragments of theligands, or otherwise are varied in accord with conventional practice.

The Ka, or affinity constant, with which the ligand binds to receptor isthe ratio of the rate at which the ligand binds to receptor (the "on"rate) divided by the rate at which the ligand disassociates from thereceptor at the site in question (the "off" rate). A high affinityinteraction is one in which the "on" rate predominates Antagonistsgenerally will have high affinity variation at site 1. For the most parta high affinity variant at either site is desirable for agonistsdepending on the nature of the receptor. If the ligand binds to thereceptor and the ternary receptor complex issues a single rather thancontinuous signal, a ligand analogue having an extremely high affinityfor receptor may in fact occupy receptor for so long that it, in effect,begins to act as an antagonist. For example, if 20-30 minutes arerequired for the dimerized receptor to signal a change in the characterof the cell (e.g., releasing protein, stimulating mitosis, etc.), thenit would be unnecessary for an agonist ligand to possess a rate constantwhereby it occupies the receptor for a matter of hours. Thus, theagonist affinity optimally should be optimized so that the variantdissociates from the receptor after approximately the same period as isrequired to complete the receptor signalling event. A high (>fold overwild type) site 1 Ka is entirely desirable for an antagonist having aninactive site 2 since this would enhance the occupation of receptor site1 by the antagonist and thereby tie up receptor that otherwise mightbecome available to native ligand. Thus, agonist mutations mostdesirably will have rate constants consistent with the signallingcharacter of the dimerized receptor, while antagonists will exhibit highaffinity at site one and lower (or absent) affinity at site two. Ka isreadily determined for a given ligand variant, for example by use ofBIA-core equipment which is commercially available from Pharmacia.

The ability of ligand and its receptor to form ternary complex alsoserves as a convenient assay endpoint for substances that influence theformation of the complex but which are not ligand amino acid sequencevariants. For example, if one desired to screen a group of candidatenon-peptidyl or short peptide molecules for agonist or antagonist effectone need only follow the formation of the ternary complex in thepresence and in the absence of the candidate. Candidates that suppresscomplex formation will act as antagonists; those that reduce thequantity of ligand and receptor required to form the complex will beagonist candidates. Non-specific effects on the receptor or ligand, e.g.protein denaturation, are excluded by conventional analysis, e.g. CDstudies.

Similarly, the assay method is useful to detect variant receptors andtheir activity. For example, a mutant receptor is assayed for itsability to bind correctly to ligand by measuring its ability to competewith native receptor for a ligand binding site. In such an assay usinghomoquenching, fluorescein labeled receptor is added to a limitingamount of native ligand, and the ability of the candidate receptor tocompete with labeled receptor is measured by increased fluorescence inrelation to receptor standard. Fluorescence quenching or enhancementalso can be detected by labeling half of the receptor population withone fluorophore and the other half with the enhancing or quenchingmolecule. Such systems are widely known and are generally applicable tothe ternary assay. The assay also can be adapted to permit analysis ofsite 1 or site 2 binding by simply following the molecular size of thecomplex. If the ligand and receptor do not form any complex at all,despite adequate proportions, then site 1 is deficient, or (whenreceptor is the candidate) the receptor binding site for ligand site 1is deficient. If only a 1:1 complex is formed (even though adequateamounts of ligand or receptor are present) then ligand site 2 (or itsreceptor site, depending upon the candidate used) is deficient in itsability to bind to receptor. The same analysis is applied to identifyingligands or receptors that are capable of binding at site 1 or 2 withgreater affinity than the wild type protein.

Assay For Complex Formation

Assay methods for detection of the ternary complex include determiningthe molecular weight of the complex, determining fluorescence emissionor fluorescence quenching or other energy transfer between labels on thereceptor, Bia-core analysis, gel exclusion chromotography, native gelelectrophoresis, isoelectric focusing, sedimentation and dialysis. Othersuitable methods include the use of antibodies binding to thereceptor-ligand sites, optical rotation of polarized light,chromatography, and nuclear magnetic resonance. Among the types ofchromotography are gel filtration, ion exchange and high pressure liquidchromotography (HPLC). Any method of analysis will work that allows adetermination of the ternary complex formation against a background ofthe uncomplexed ligand and/or receptor.

Ligands and their Receptors

Included among the ligands which are structurally analyzed and, ifappropriate, mutated in accord herewith are growth hormones,insulin-like growth factors, parathyroid hormone, insulin, relaxin,glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and leutinizing hormone(LH),hemopoietic growth factor, hepatic growth factor, fibroblast growthfactor, prolactin, placental lactogen, tumor necrosis factor-alpha and-beta, mullerian-inhibiting substance, mouse gonadotropin-associatedpeptide, inhibin, activin, vascular endothelial growth factor,integrins, thrombopoietin, nerve growth factors such as NGF-β,platelet-derived growth factor, transforming growth factors (TGF) suchas TGF-alpha and TGF-beta, insulin-like growth factor-I and -II, EPO,osteoinductive factors, inteferons such as interferon-alpha, -beta, and-gamma, colony stimulating factors (CSFs) such as M-CSF, GM-CSF, andG-CSF, interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8 and other polypeptide factors. Preferred ligands are helicalmonomeric cytokines/hormones such as G-CSF. GM-CSF, II-2, IL-3, IL-4,IL-6, IL-7, EPO, growth hormone, placental lactogen and prolactin.Receptors for these ligands are used in the same fashion as for hGH andantagonists or agonist are selected as described above.

The foregoing discussion has concentrated on amino acid sequencevariation. However, the same objectives also are accomplished byconvalently modifying the target residues(s) by in vitro methods. Thismay be effected through any type of chemical modification which disruptsor modifies the ability of the side chains of the residues at the targetlocations to bind to receptor. The net effect is the same, e.g., assubstitutional mutations provided the covalent modification issufficiently specific for the target residue(s). Specificity is achievedby selecting agents which react preferentially with the desired sidechain: additional specificity is achieved by blocking other side chainswith antibodies which bind to the regions to be protected. Themodification may be of one or more of the amino acids that directlyparticipate in the binding (structural residues); alternatively aminoacids adjacent or in the region of receptor binding which are involvedin maintenance of conformation are covalently modified in vitro. Thecovalent modification includes such reactions as oxidation, reduction,amidation, deamidation, condensation and so forth, or substitution ofbulky groups such as polysaccharides or polyethyleneglycol. Methods forcovalently attaching such moieties (e.g., polyethyleneglycol) toproteins are well known (see for example Davis, et al. U.S. Pat. No.4,179,337).

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful: the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing amino-containing residues include imidoesterssuch as methyl picolinimidate; pyridoxal phosphate; pyridoxal;chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea;2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate,and N-hydroxysuccinamide esters of polyethylenene glycol or other bulkysubstitutions.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵ I or ¹³¹ I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R'--N═C═N--R'), where R and R' aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Evidence for two binding sites on the hGH comes from antibody binding.The affinity of hGH for the hGHbp is measured by displacement of [¹²⁵I]hGH from the hGHbp and precipitating the complex with an anti-receptormonoclonal antibody (Mab5) produced from glycosylated rabbit GH receptor(Barnard, R. et al., Endocrinology 115:1805-1813 (1984); Barnard, R. etal., Biochem. J. 231:459-468 (1985)). Using this assay, Scatchardanalysis shows that hGH and the hGHbp form a 1:1 complex (Leung, D. W.et al., Nature 330:537 (1987); Fuh, G. et al., J. Biol. Chem.265:3111-3115 (1990) Barnard, R. et al., Endocrinology 115:1805-1813(1984): Barnard, R. et al., Biochem. J. 231:459-468 (1985); Spencer, S.A., et al., J. Biol. Chem., 263:7862-7867 (1988)). Scatchard analysis ofdisplacement curves (FIG. 3), using another set of MAbs (MAb387 or 3D7),produced stoichiometries of 0.5 hGH to 1 hGHbp. These results can beexplained if Mab5 were to block determinants on the hGHbp for binding toa second site on hGH.

Evidence for two binding sites on each hGH polypeptide was developedusing monoclonal antibodies in the scanning-mutational analysis ofbinding determinants between hGH (Cunningham, B. C. et al., Science,244:1081 (1989)) and the hGHbp (Example 2). The determinants identifiedin these studies are important for modulating formation of the 1:1complex. Based upon this data we installed these determinants intonon-binding homologs of hGH and created analogs that bind tightly to thehGHbp (Cunningham, B. C., et al., Science, 247:1461-1465 (1990)). Byincorporating 8 substitutions into hPRL or 5 into hPL, we have producedvariants that bind to the hGHbp and form a 1:1 complex.

We used gel filtration to determine if these hPRL or PL variants couldbind to one or two molecules of the hGHbp. At a 2:1 ratio of hGHbp tohormone, both binding variants of hPRL show two symmetrical peakscorresponding to a 1:1 complex with the hGHbp.

We employed scanning calorimetry to further evaluate the stoichiometryand heat of reaction because binding can be studied free in solutionwithout the need to employ antibodies or chromatography to separatecomplexes (FIG. 5). This experiment allowed us to determine theequivalents of hGH bound to the hGHbp and the heat of reaction.

In Table 1d we summarize the titration end points for wild-type hGH andvariants of hGH and hPRL. The ratio of hGH necessary to bind to all ofthe hGHbp is about 0.5 to 1. Altogether these data strongly indicatethat the hPRL and hPL variants are missing important determinants fordimerization of the hGHbp. These determinants are largely conserved inthe single alanine mutants of hGH but not in the hPRL or hPL variants.This suggests there are two binding sites on hGH for the hGHbp. One ofthese sites has been functionally characterized in detail byalanine-scanning mutagenesis of hGH (Cunningham B. C. et al., Science,244:1081 (1989)) or the hGHbp using the Mab5 or Mab263immunoprecipitation assay, respectively. The second sites on hGH and thehGHbp remained to be elucidated.

Binding of hGH to the hGHbp causes little spectral change in thecomponents. We investigated the change in the circular dichroic (CD) andfluorescence spectra upon complex formation. When hGH and the hGHbp aremixed the far UV CD spectrum is virtually identical to the sum of thespectra of hGH and hGHbp (FIG. 6A). This result indicates the absence oflarge changes in regular secondary structure upon formation of thecomplex. The near UV CD spectrum (FIG. 6B) reflects the asymmetricenvironment of the aromatic amino acid side chains (Bewley, T. A. RecentProgress in Hormone Research, 35:1555 (1979); Bewley T. A. et al.,Archives of Biochemistry and Biophysics, 233:219-227 (1984)). There arelarge differences between the UV absorbance spectra of hGH and thehGHbp, largely a result of the greater tryptophan content of the hGHbpcompared to hGH (9 versus 1, respectively). However, except for anincrease in the intensity of the spectrum the sum of the individualspectra are essentially identical to that obtained after mixing.

In the fluorescence spectrum, there is a blue shift from 340 nanometersto 334 nanometers and slight reduction in the fluorescence intensityupon hGH binding to the hGHbp (FIG. 7). Iodide quenching andStern-Volmer analysis indicate there is a reduction in the exposure oftryptophan in the hormone receptor complex. This is likely the result ofburying one or more Trp residues in the hGHbp upon binding hGH becausefluorescence quenching studies have shown that the tryptophan in hGH isnot appreciably exposed to solvent (Bewley, T. A., Recent Progress inHormone Research, 35:1555 (1979); Bewley, T. A. et al., Archives ofBiochemistry and Biophysics, 233:219-227 (1984)). In contrast,mutational analyses of the hGHbp show that Trp104 is especiallyimportant in binding to hGH.

As discussed above, the hGH results are relevant to other polypeptideligands, e.g., hormone-receptor and cytokine-receptor systems. Thegrowth hormone and prolactin receptors appear to be structurally relatedto a large family of cytokine receptors for interleukin 2, 3, 4, 5, 6,7, erythropoetin, macrophage colony stimulating factor and others (forreviews see 8). It is striking that the intracellular domains of thesereceptors share little if any sequence homology, and none appearhomologous to any known tyrosine kinase. Nonetheless, the GH (Carter-Su,C., et al., J. Biol. Chem., 264:18654-18661 (1989)), IL-2 (Asao, H., etal., J. Exp. Med. 171:637-644 (1990)), and IL-3 (Itoh N., et al.,Science 247:24-327 (1990)) receptors become phosphorylated shortly afterhormone binding. In the case of the IL-2 (Sharon, M. et al., Proc. Natl.Acad. Sci. U.S.A. 87:4869-4873 (1990)) and IL-6 (Taga, T., et al., Cell58, 573-581 (1989)) receptors there is evidence indicating thataccessory proteins and/or receptors are involved in signal transduction.The present results with hGH and its binding protein, support a modelfor activation of the hGH receptor in which hGH binding inducesdimerization of the extracellular portion of the receptor which bringstogether the intracellular domains to create an active domain that mayinteract with cytoplasmic (or membrane bound) components. This may ormay not occur without substantial change in conformation of thecomplexed components.

Two other groups have recently crystallized a polypeptide hormone incomplex with its extracellular binding domain (Lambert, G., et al., J.Bio. Chem. 264:12730-12736 (1989); Gunther, N., et al., J. Biol. Chem.265:22082-22085 (1990)); however neither reports conclusive evidence forreceptor dimerization. Human IL-2 was crystallized predominantly in a1:1 complex with a soluble recombinant form of the human p55 componentof the IL-2 receptor, although a small amount of disulfide linked p55dimer was observed. Cross-linking studies suggest that the functionalIL-2 receptor complex is a heterodimer formed between IL-2, p55 andanother receptor component called p70 (Saragori H., et al., J. Immunol.139:1918-1926 (1987); Ogura, T., et al., Mol. Biol. Med. 5:123-138(1988)). The extracellular domain of the EGF receptor (EGFbp) has beencrystallized in complex with one molecule of EGF (Gunther, N., et al.,J. Biol. Chem. 265:22082-22085 (1990)). Binding studies andsedimentation analysis indicate the formation of a 1:1 EGF. EGFbpcomplex in solution. These data suggested that the extracellular domainis insufficient to undergo hormone induced dimerization. However, it isnoteworthy that the binding studies used anti-EGF receptor polyclonalantibodies to precipitate the complex. Furthermore, the crystallizationand sedimentation experiments used a large excess of hormone overreceptor. In our case, Mabs raised against the natural GH receptor blockdimerization (FIG. 3) and a large excess hGH will dissociate thehGH.(hGHbp)₂ complex into a monomeric complex (FIG. 2, Ref. 34).

This later effect may have important pharmacologic implications. hGH isnaturally produced in pulses that exceed 5 nM in serum and levels dropquickly to well below 1 nM (Taylor, A. L., et al., J. Clin. Invest.48:2349 (1969); Thompson, R. G., et al., J. Clin. Invest. 51:3193(1972); Ho, K. Y., et al., J. Clin. Endocrinol. Metab. 64:51-58 (1987)).However, the hGHbp is present naturally in serum at a constant level ofabout 0.5 to 1 nM (Baumann, G., et al., J. Clin. Endocrinol. Metab.62:134-141 (1986); Herington, A. C., et al., J. Clin. Invest.77:1817-1823(1986)). Thus, as hGH is pulsed in excess over the hGHbp onewould expect it to produce 1:1 complexes with the hGHbp as well as freehGH that could interact with cellular receptors (even producingheterodimeric complexes having the form hGH.hGHbp.hGHmembrane-receptor).

We have determined that hGH interacts with hGHbp to make a complex ofthe form hGH(hGHbp)₂ and have proposed that the resulting dimerizationof the extracellular receptor domain initiates somatogenic signaltransduction for this hormone. Since the recruited hPL and hPRL analogs(Example 4) do not promote hGHbp dimerization we can conclude that thehPL and hPRL scaffolds lack necessary dimerization determinants whichare distinct from those required for receptor recognition and binding.To localize the domains involved, a series of hGH mutants with hPL orhPRL homolog substitutions, and two deletion analogs, were screened forreductions in hormone induced receptor dimerization. Important sidechains were then identified by a more detailed alanine-scanningstrategy.

A hGHbp variant (S237C) was constructed and fluorescently labeled.Fluorescence quenching was measured to monitor hormone induceddimerization as shown in FIG. 8. This quenching indicated a 1:2 molarratio of hGH to hGHbp (Example 4). A series of homolog-scan hGH variantswith hPL and hPRL segment substitutions were tested in the fluorescenceassay (Table 2). Four of these caused significant reductions in hormoneinduced hGHbp dimerization (Example 4). In the other two hGH deletionanalogs, the loss in hGHbp dimerization appears to be due to disruptionsin secondary site hGHbp binding (Table 2).

The mutant hGHbp (S237C-AF) was fluorescently labeled. This fluorescentsignal was used to monitor hormone induced dimerization as shown in FIG.8. The hGH is serially diluted against a fixed 10 nM concentration ofS237C-AF and fluorescence quenching measured at equilibrium.Homoquenching of the fluorescein label increases with hGH addition andbecomes maximal at 0.5 molar equivalents of hGH However, quitestrikingly, this homoquenching is reversed at higher concentrations ofhGH indicating hGH.(hGHbp)₂ dissociates to hGH.hGHbp monomeric complexin the presence of excess hGH.

A series of homolog-scan hGH mutants with hPL and hPRL segmentsubstitutions were tested in the S237C-AF based assay (table 2). Threeof these, hPRL(12-19), hPRL(54-74) and hPRL(111-109) caused significantreductions (18, 6 and >100 fold). Losses in primary site (site 1 )binding for these mutants appear to largely account for the observedreductions in hGHbp dimerization. Furthermore, mutations of primary sitedeterminants (e.g. R64A and K172A/F 176A) which reduce binding affinityhave also been shown to reduce dimerization and an hGH mutant (E174A)shown to enhance hGHbp affinity for the primary site also enhancesdimerization as measured in our assay. In addition to the homolog-scanmutants, an hGH deletion analog (deletion 1-8) showed a dramaticreduction (>100 fold) in ability to induce hGHbp dimerization (Example4). This loss in hGHbp dimerization also appeared to be due todisruptions in secondary site hGHbp binding.

Specific amino acid specific side chains involved in secondary sitehGHbp binding were probed by alanine scanning (Example 6). An analysisof 26 alanine mutants (table 3) revealed only two mutants, F1A and I4Awhich cause >10-fold disruptions in hGHbp dimerization and 4 others(L6A, R8A, D116A, E119A) carrying >2-fold disruption. These determinantsare different from those crucial for primary site binding; Experimentsin which sequential hGH additions are made to a fixed concentration ofS237C-AF (100 nM), and fluorescence homoquenching showed rapidequilibration times (<3 minutes) for hGH induced dimerization and slowequilibration times (>30 minutes) for subsequent reversal ofdimerization by excess hGH. This suggests that reversal of dimerizationis off-rate limited (Example 6 for mechanism).

The formation of hGH(hGHbp)₂ crystals permits the determination of thethree-dimensional structure of the hGH(hGHbp)₂ complex using x-raychrstallographic techniques following the methods described in Blundelland Johnson, Academic Press, London, 1976. This structure is illustratedin FIG. 11 and discussed in Example 7 below. The structure of FIG. 11indicates that each hGH is bound to two hGH receptors, or hGHbp. EachhGHbp is in contact with different portions of the hGH; the hGH aminoacids in contact with the first hGHbp are shown in Table 4; and, the hGHamino acids in contact with the second hGHbp are shown in Table 5. Thecontacting amino acids between the two hGHbp are shown in FIG. 6.

Variants of hGH can be made at these amino acid contact points anddetected by the assay method of the present invention. hGHbp variantssimilarily can be made in those amino acids involved in the binding tothe hGH or between the two hGHbp themselves. Such hGHbp variants candetected using the assay methods of the present invention using wildtype hGH and hGHbp.

Therapeutic Compositions and Administration of Ligand Analogs and GHBPAntibodies

Therapeutic formulations of ligand analogues or GHbp antibody areprepared for storage by mixing the ligand analogues protein having thedesired degree of purity with optional physiologically acceptablecarriers, excipients, or stabilizers (Remington's PharmaceuticalSciences, supra), in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides (to prevent methoxide formation);proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, Pluronics or polyethylene glycol (PEG).

Ligand analogues or GHGp antibody to be used for in vivo administrationmust be sterile. This is readily accomplished by filtration throughsterile filtration membranes, prior to or following lyophilization andreconstitution. Ligand analogues or antibody to a ligand analoguesordinarily will be stored in lyophilized form or in solution.

Therapeutic ligand analogues, or ligand analogues specific antibodycompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The route of administration of ligand analogues or GHbp antibody is inaccord with known methods, e.g., injection or infusion by intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional routes, or by sustained release systemsas noted below. Ligand analogues are administered continuously byinfusion or by bolus injection. GHbp antibody is administered in thesame fashion, or by administration into the blood stream or lymph.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles. e.g. films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels [e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer, et al., J. Biomed. Mater. Res., 15:167-277 (1981)and Langer, Chem. Tech., 12:98-105 (1982) or poly(vinylalcohol)],polylactides (U.S. Pat No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman, et al.,Biopolymers, 22:547-556 [1983]), non-degradable ethylene-vinyl acetate(Langer, et al., supra), degradable lactic acid-glycolic acid copolymerssuch as the Lupron Depot™ (injectable micropheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(-)-3-hydroxybutyric acid (EP 133,988). While polymers such asethylene-vinyl acetate and lactic acid-glycolic acid enable release ofmolecules for over 100 days, certain hydrogels release proteins forshorter time periods. When encapsulated proteins remain in the body fora long time, they may denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor protein stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS--S bond formation through thio-disulfide interchange, stabilizationmay be achieved by modifying sulfhydryl residues, lyophilizing fromacidic solutions, controlling moisture content, using appropriateadditives, and developing specific polymer matrix compositions.

Sustained-release ligand analogues or antibody compositions also includeliposomally entrapped ligand analogues or antibody. Liposomes containingligand analogues or antibody are prepared by methods known per se: DE3,218,121; Epstein, et al., Proc. Natl. Acad. Sci. U.S.A., 82:3688-3692(1985); Hwang, et al., Proc. Natl. Acad. Sci. U.S.A., 77:4030-4034(1980); EP 52,322: EP 36,676; EP 88,046; EP 143,949: EP 142,641;Japanese patent application 83-118008: U.S. Pat. No. 4.485,045 and U.S.Pat. No. 4.544,545: and EP 102,324. Ordinarily the liposomes are of thesmall (about 200-800 Angstroms) unilamelar type in which the lipidcontent is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal ligand analogues therapy.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No5,013,556.

Use of Variants

Antagonist ligand variants selected by the assay of the presentinvention are used in therapeutic formulations or expressed intransgenic animals to act as antagonists, blocking the action of thenaturally occuring ligand. Transgenic animals are useful as novelties oras experimental models. Other selected ligand variants are used intherapeutic formulations to act as agonists, administered to potentiateor to promote a response similar to that stimulated by the naturallyoccuring cytokine. For example, hGH variants are used in apharmaceutically effective dosage formulation (e.g., U.S. Pat. No.5,096,885 filed Apr. 15, 1988). Ligand variants are advantageous in thatthey may have an activity similar to the naturally occuring cytokine,but with reduced or eliminated undesirable side effects, one suchexample being an hGH variant that does not exhibit diabetogenicactivity. Ligand variants which have no biological activity, either asagonists or antagonists, are useful in immunoassays for the wild typeligands or their antibodies since they will retain at least one ligandimmune epitope.

Monoclonal Antibody and Stimulation of Receptors

We have determined that certain antibodies are capable of stimulatingthe hGH receptor, i.e., they are capable of crosslinking the receptorsin a fashion that mimics the ability of hGH to form a ternary complexand activate the receptor. Examples of such agonist antibodies werealready known at the time of this invention, but their ability to act asagonists of hGH was unappreciated. Suitable antibodies are MAb 263(Barnard, et al., Endocrinology, 115:1805-1813 [1984] or Barnard, etal., Biochem. J., 231:459-468 [1985]). Others are MAbs 13E1 and 3D9,produced by methods described below. These antibodies optionally areused to create chimeras or CDR grafted forms that are less immunogenicthan the parental antibodies in the intended host. The antibodiespreferably are directed against the human receptor. Agonists for hGHmust be at least bivalent. However, monovalent antibodies such as FAbfragments, which only bind to one receptor molecule, are useful asantagonists.

The bivalent antibodies are bispecific in some embodiments. Thus, onearm of the antibody is directed against one receptor epitope while theother arm is directed against another epitope on the receptor.Antagonist antibody embodiments can contain one arm directed to thereceptor (preferably its receptor-receptor contact region) with anotherarm directed at an antigen other than GH receptor. The antibodies aremade by conventional hybridoma methods or by conventional hydridomamethods or by recombinant methods wherein the recombinant cell istransformed with heavy and light chain encoding each arm. Bispecificantibodies are made by recombinant methods and recovered by affinitypurification from the cell culture, or the antibodies are madeseparately and recombined in vitro by conventional methods.

These results are of particular interest in the veterinary field sinceit is now possible to raise such antibodies in vivo by immunizing theanimals against the growth hormone receptor or fragment thereof so as togenerate GH agonists by active immunization. Thus, antibodies areadministered either passively (by administration of exogenous antibody)or actively by immunization with receptor.

The agonist antibodies are administered in dosages based on theiraffinity in comparison to growth hormones. Further dosages for mammalsare readily extrapolated from the rat growth study described infra.Antagonist antibodies are administered in dosages calculated to completewith sufficient growth hormone to reduce the effective activity thereofto normal ranges or to below normal if dwarf animals are the objective.

The antibodies are formulated and administered in substantially the samefashion as the ligand analogues as described above. The agonistantibodies are used for the same purposes as growth hormone has beenused heretofore.

The following examples are intended to illustrate the best mode nowknown for practicing the invention, but the invention is not to beconsidered limited to these examples.

EXAMPLE 1 STRUCTURE OF THE hGH-RECEPTOR COMPLEX

The assay methods of the present invention are based upon the discoveryof the hGH-receptor complex structure; that is, one hGH and two hGHreceptors or binding proteins forming a stable complex that may bedetected. These assay methods are exemplified by the hGH(hGHbp)₂ complexassay methods.

Crystallization of the hGH.(hGHbp)₂ Complex

Crystals of the complex between hGH (22 kDa) and the hGHbp (28 kDa)(FIG. 1a) were grown by vapor phase diffusion (A. McPherson, inPreparation and Analysis of Protein Crystals, John Wiley and Sons, NewYork, (1982)). The crystals diffract to at least 2.7 Å and belong tospace group (P2₁ 2₁ 2) with unit cell parameters of a=145.8 Å, b=68.6 Å,c=76.0 Å. The volume of the asymmetric unit of these crystals is suchthat the complex is unlikely to have the form of either hGH.hGHbp or(hGH.hGHbp)₂. In particular, the solvent content would have to be toohigh (68%) for a 1:1 complex, or too low (32%) for a 2:2 complex in theunit cell. Since the typical solvent content of crystals is about 50%(Matthews, B. W. J. Mol. Biol. 33, 491-497 (1968)) it was most likelythat these crystals contain an asymmetric mixture of the components.

To evaluate the precise composition of the crystals they weredissociated in 0.1% trifluoroacetic acid and chromatographed underdenaturing conditions (FIG. 1b). The amount of hGH and hGHbp wasquantified by integration of their respective peaks that were monitoredat 214 nm, which corresponds to the absorbance of peptide bonds. Fromfour independent determinations, the ratio of the A₂₁₄ of the hGH peakto the hGHbp peak was 0.42±0.02. For a complex having the formhGH.(hGHbp)₂, the ratio predicted for integrated peak areas is 0.40based upon the number of residues in each of the components (191residues for hGH and 238 residues for the hGHbp). In controlexperiments, a 1:2 mixture of hGH to the hGHbp produced essentially thesame chromatogram as FIG. 1B whereas 2:1 and 1:1 mixtures generatedexpected and different chromatograms. Therefore, the crystals in FIG. 1contained hGH and hGHbp in a 1:2 molar ratio. The ability of hGH andhGHbp to form a stable complex in solution confirms that complexformation is a reliable assay parameter.

Formation of the hGH (hGHbp)₂ Complex in Solution

The existance of the hGH (hGHbp)₂ complex was established in solution bysize exclusion chromatography. hGH and the hGHbp were mixed in ratios of1:4, 1:3, 1:2, 1:1 and 1:0.5 and the components were separated by gelfiltration on a Superose 12 FPLC column (FIG. 2). At a 1:4 ratio of hGHto hGHbp two peaks are present of apparent molecular weight 70 kD and 30kD corresponding to a hGH.(hGHbp)₂ complex and free hGHbp, respectively.The areas of the peaks are dominated by the absorbance of the hGHbpbecause its ε₂₈₀ ⁰.1% is 2.9-fold higher than hGH 12. The ε₂₈₀ ¹.0% forhGH is 0.82 cm⁻¹ and 2.35 cm⁻¹ for the hGHbp based on absorbance andcompositional analysis of a pure sample. At 1:3 and 1:2 ratios of hGH tohGHbp there is no change in the shape or position of the complex peak;however the peak corresponding to the free hGHbp is progressivelyreduced to zero. Thus at a 1:2 ratio, virtually all of the hGH and hGHbpare bound in a complex. As the ratio of hGH to hGHbp is adjusted to 1:1and finally 1:0.5, the position of the complex peak shifts to a smallersize (˜55 kD), becomes asymmetric, and free hGH accumulates, therebysuggesting there is a mixture of species corresponding to hGH.(hGHbp)₂,hGH.hGHbp and monomeric hGH. SDS-PAGE of protein samples taken acrossthese peaks confirmed the assigned compositions. Additional controlexperiments showed that the free components run as monomeric proteinsindicating that dimerization requires the presence of both hGH and thehGHbp under these conditions. Therefore, complex formation is detectableby multiple assay methods and hGH.(hGHbp)₂ complex formation serves asan indicator of hGH binding to cellular receptors. Similarly, anycytokine acting through a cytokine receptor which forms acytokine-cytokine receptor complex, analogous to the hGH--hGH receptorcomplex, can be evaluated by such assay procedures.

EXAMPLE 2 hGH RECEPTOR BINDING SITES

The nature of hGH binding sites for hGH receptor or hGH binding proteinwas characterized using antibody that blocked hGH binding sites for thereceptor or binding protein. The evidence for two binding sites on thehGHbp is as follows.

The affinity of hGH for the hGHbp is typically measured by displacementof [¹²⁵ I]hGH from the hGHbp and precipitating the complex with ananti-receptor monoclonal antibody (Mab5) produced from glycosylatedrabbit GH receptor (Barnard, R. et al., Endocrinology 115:1805-1813(1984); Barnard, R. et al., Biochem. J. 231:459-468 (1985)). Using thisassay, Scatchard analysis demonstrated that hGH and the hGHbp werecapable of forming a 1:1 complex (Leung D. W. et al., Nature 330:537(1987); Fuh G., et al., J. Biol. Chem. 265:3111-3115 (1990); Barnard.R., et al., Endocrinology 115, 1805-1813 (1984); Barnard, R., et al.,Biochem. J. 231:459-468 (1985); Spencer, S. A., et al., J. Biol. Chem.263:7862-7867 (1988). Spencer, S. A., et al., J. Biol. Chem.263:7862-7867 (1988)).

Recently, additional Mabs have been produced by immunization with theunglycosylated hGHbp purified from E. coli (Fuh G., et al., J. Biol.Chem. 265:3111-3115 (1990)). Scatchard analysis of displacement curves(FIG. 5) using two of these anti-hGHbp Mabs (3B7 and 3D9) to precipitatethe complex give higher binding affinities (K_(D) 0.1 nM versus 0.4 nMfor Mab5) and stoichiometries of 0.5 hGH to 1 hGHbp. These results canbe explained if Mab5 were to block determinants on the hGHbp for bindingto a second site on hGH. The lack of cooperativity in assays usingMab3B7 and 3D9 is likely to reflect the fact that the affinity of hGH inthe 1:1 complex (as measured using Mab5) is only about four-fold weakerthan for the 1:2 complex (as measured using Mab 3B7 and 3D9). A muchgreater differential affinity would be needed to pick up positivecooperativity by upward inflections on a Scatchard plot. Moreover, Mab3B7 and 3D9 should precipitate both 1:1 and 1:2 complexes which woulddampen any apparent cooperativity. Therefore, blockage of the hGH secondbinding site results in a 1:1 hGH-hGHbp molar ratio: while antibody thatdoes not block the second binding site results in a 1:2 molar ratio.

The evidence for two binding sites on hGH is the following. Mab5 wasemployed in the scanning-mutational analysis of binding determinantsbetween hGH and the hGHbp. Therefore, the determinants identified inthese studies reflect those important for modulating formation of the1:1 complex. Based upon this data we have installed these determinantsinto non-binding homologs of hGH and created analogs that bind tightlyto the hGHbp (Cunningham, B. C., et al., Science 247:1461-1465 (1990)).For example, wild-type human prolactin (hPRL) or human placentallactogen (hPL) bind to over 10⁵ or 10³ -fold more weakly to the hGHbpthan does hGH, respectively. By incorporating 8 substitutions into hPRL(E62S/D63N/Q66E/H171D/E174A/N175T/Y176F/K178R; (Cunningham, B. C., etal., Science 247:1461-1465 (1990)) or 5 into hPL(V4L/D56E/M64K/E174A/M179I); we have produced variants that bind to thehGHbp only 6.2- or 1.4-fold weaker than hGH, respectively.

We used gel filtration to determine if these variants could bind to oneor two molecules of the hGHbp (FIG. 3). At a 2:1 ratio of hGHbp tohormone, both binding variants of hPRL and hPL show two symmetricalpeaks corresponding to a 1:1 complex with the hGHbp (apparent molecularweight of about 55 kDa) and a lower molecular weight peak (30 kDa)representing a stoichiometric excess of the hGHbp. Under identicalconditions, the wild-type hGH produces a single peak (apparent molecularweight 77 kDa) corresponding to the hGH (hGHbp)₂ complex. The smallsatellite peak is from a slight excess of hGHbp. Peak compositions wereconfirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

We employed scanning calorimetry to further evaluate the stoichiometryand heat of reaction because binding can be studied free in solutionwithout the need to employ antibodies or chromatography to separatecomplexes. To a solution containing a fixed concentration of the hGHbp(15 μM), aliquots of hGH were added and the heat of reaction wasmeasured until there was no further enthalpic change (FIG. 5). Thisexperiment allowed us to determine the equivalents of hGH bound to thehGHbp and the heat of reaction.

In Table 14 we summarize the titration end points and heats of reactionfor wild-type hGH and variants of hGH and hPRL. The ratio of hGHnecessary to bind to all of the hGHbp is about 0.5 to 1. Furthermore, aseries of single alanine mutants that reduce binding (by up to 20-fold)or enhance binding (by 4.5-fold) give the same stoichiometry of bindingas wild-type hGH to the hGHbp albeit with changes in the enthalpy of thereaction. In contrast, the stoichiometry of binding of the hPRL variantto the hGHbp is 0.85 to 1.

                  TABLE 1d                                                        ______________________________________                                        Stoichiometries and heats of reaction with the hGHbp for                      wild-type and alanine mutants of hGH and a variant of                         hPRL that binds tightly to the hGHbp.                                                               mol hGH/mol GHbp                                        Protein     K.sub.D (nM).sup.a                                                                      at the end point.sup.b                                  ______________________________________                                        wt hGH      0.34      0.46 ± 0.05                                          R64A        7.1       0.47 ± 0.07                                          K172A       4.6       0.48 ± 0.1                                           I58A        5.6       0.48                                                    F176A       5.4       0.46                                                    E174A       0.08      0.47                                                    hPRL variant                                                                              21        0.85                                                    ______________________________________                                         .sup.a Values taken from Cunningham B.C., et al., Science 244:1081 (1989)     for formation of a 1:1 hGHhGHbp complex using the Mab5 immunoprecipitatio     assay. Calorimetry is not suitable for measuring binding constants for th     dimeric complex in the nanomolar range because the calorimeter is not         sensitive to enough to accurately determine the change in the heat of         reaction for components whose concentrations would need to be set below       the binding constant.                                                         .sup.b Average (±SE) of duplicates; others were single determinations.

Altogether these data strongly indicate that the hPRL and hPL variantswere missing important determinants for dimerization of the hGHbp. Thesedeterminants are largely conserved in the single alanine mutants of hGHbut not in the hPRL or hPL variants. This suggests there are two bindingsites on hGH for the hGHbp. One of these sites has been functionallycharacterized in detail by alanine-scanning mutagenesis of hGH(Cunningham B. C., et al., Science 244:1081 (1989)) or hGHbp using theMab5 immunoprecipitation assay, respectively. The second sites on hGHand the hGHbp remained to be elucidated and are described infra.

EXAMPLE 3 hGH-RECEPTOR COMPLEX AND SPECTRAL CHANGE

Binding of hGH to its receptor causes little spectral change in thecomponents. To determine if the binding of hGH to the hGHbp causes largechanges in secondary or tertiary structure of the components weinvestigated the change in the circular dichroic (CD) and fluorescencespectra upon complex formation. Proteins were prepared for spectroscopyby dialyzing approximately 1.0 mg/ml protein in 0.01M Tris pH (8.0) and200 mM NaCl. After dialysis the solutions are filtered (0.22μ,Millipore/and the absorbance spectrum was obtained. The spectra werecorrected for light scattering (Shauenstein E., et al., J. Polymer Sci.16:45 (1955)) and protein concentrations were determined by absorbanceat 280 nm. The ε₂₈₀ ⁰.1% for hGH is 0.82 cm⁻¹ and 2.35 cm⁻¹ for thehGHbp based on absorbance and compositional analysis of a pure sample.hGH exhibits a strongly co-helical CD spectrum (Bewley T. A., et al.,Arch. Biochem. Biophys. 138:338-346 (1970)) characteristic of its 4helix bundle structure (Abdel-Meguid S. S., et al., Proc. Natl. Acad.Sci. U.S.A. 84:6434 (1987)). In contrast, the CD spectrum of the hGHbpis characteristic of a protein composed mainly of turns and loops(Cleary, S., et al., Biochemistry 28:1884 (1989): Hilder, R. C., et al.,Biophysical Chemistry 31:45 (1988)) connected by disulfide bonds; thehGHbp contains 3 adjacently linked disulfide bonds (Fuh G., et al., J.Biol. Chem. 265:3111-3115 (1990)). Frozen cell paste was thawed inhypotonic buffer (10 mM Tris pH 8.0, 1 mM PMSF (Sigma), 2 mM EDTA). Thesuspension was homogenized, stirred for 1 hr at 4° C., and thencentrifuged at 10,000×g for 20 min. To the supernatant was added solidammonium sulfate at 260 g/L and stirred until dissolved. The proteinprecipitate was collected by centrifugation at 10,000×g for 30 min. Thepellet was resuspended in 10 mM Tris pH 8.0, 1 mM PMSF, and dialyzedagainst the same buffer. The dialysate was applied to a Q Sepharosecolumn (Pharmacia) in 10 mM Tris (pH 8.0) and eluted with a lineargradient of 0.0 to 0.5M NaCl. Peak fractions containing the hGHbp wereloaded directly onto an hGH affinity column. After washing, the columnwas eluted with 4M MgCl₂, 10 mM Tris pH 7.5. The peak fractions werecombined and dialyzed with 10 mM Tris pH 7.5, applied to a Mono Qcolumn, washed and eluted in 10 mM Tris pH 7.5 with a linear gradient of0.0 to 0.2M NaCl.

When hGH and the hGHbp are mixed the far UV CD spectrum is virtuallyidentical to the sum of the spectra for hGH and hGHbp (FIG. 6A). Thisresult indicates the absence of large changes in regular secondarystructure upon complexation. The near UV CD spectrum (FIG. 6B) reflectsthe asymmetric environment of the aromatic amino acid side chains(Bewley, T. A. Recent Progress in Hormone Research 35:1555 (1979);Bewley T. A., et al., Archives of Biochemistry and Biophysics233:219-227 (1984)). There are large differences between the UVabsorbance spectra of hGH and the hGHbp, largely a result of the greatertryptophan content of the hGHbp compared to hGH (9 versus 1,respectively). However, except for an increase in the intensity of thespectrum the sum of the individual spectra are essentially identical tothat obtained after mixing.

In the fluorescence spectrum, there is a blue shift from 340 nanometersto 334 nanometers and slight reduction in the fluorescence intensityupon hGH binding to the hGHbp (FIG. 7). Iodide quenching and Stem-Volmeranalysis indicate there is a reduction in the exposure of tryptophan inthe hormone receptor complex. This is likely the result of burying oneor more Trp residues in the hGHbp upon binding hGH because fluorescencequenching studies have shown that the tryptophan in hGH is notappreciably exposed to solvent (Bewley, T. A., Recent Progress inHormone Research 35:1555 (1979); Bewley T. A., et al., Archives ofBiochemistry and Biophysics 233:219-227 (1984)). In contrast, mutationalanalyses of the hGHbp show that Trp 104 is especially important inbinding to hGH. While these spectral studies show little conformationalchange upon binding of hGH to the hGHbp, these methods are biased tostructural changes in regular secondary structure (far UV CD) andchanges in positions of aromatic groups (near UV CD and fluorescencequenching). Therefore, a high resolution structure of the complexed andfree components may still reveal conformational changes.

EXAMPLE 4 ASSAY METHOD AND MODIFIED hGH

Modified polypeptide hormones were evaluated in the assay method. A setof hGH residues (including F10, F54, E56, I58, R64, Q68, D171, K172,E174, F176, R178 and V185) are known to be important for confering highaffinity stoichiometric binding to hGHbp (WO 90/04788 ). Thesedeterminants were installed in the hGHbp-binding-incompetent hGHhomologs, human placental lactogen (hPL) and human prolactin (hPRL) soas to identify hPL and hPRL analogs that bind hGHbp tightly (Kd=l nM and6 nM, respectively). As previously discussed, hGH interacts with hGHbpto make a complex of the form hGH(hGHbp)₂. Since the recruited hPL andhPRL analogs do not promote hGHbp dimerization we can conclude that thehPL and hPRL scaffolds lack necessary dimerization determinants whichare distinct from those required for initial receptor recognition andbinding. To localize the domains involved in forming hGH(hGHbp)₂, aseries of hGH mutants with hPL or hPRL homolog substitutions, and twodeletion analogs, were screened for reductions in hormone inducedreceptor dimerization. Important side chains were then identified by amore detailed alanine-scanning strategy.

To quantitate hormone induced hGHbp dimerization we utilized a sensitiveassay measuring homoquenching of fluorescein-labeled hGHbp. A mutanthGHbp, S237C, was constructed, purified and reacted with5-iodoacetamidofluorescein (5-IAF) to yield fluorescently labeled hGHbp(S237C-AF). The resulting S237C-AF reagent possesses one label per hGHbpmolecule and retains full binding activity in a competitive bindingassay. Since fluorescein has excitation and emission spectra whichoverlap, this fluorescent probe undergoes homoquenching as thesemolecules approach one another. This signal was used to monitor hormoneinduced dimerization of S237C-AF as shown in FIG. 8.

In FIG. 8, homoquenching of 10 nM S237C-AF by serial addition of hGH isshown. S237C-AF was diluted to 10 nM concentration in binding buffer (20mM tris.HCl pH 7.5, 0.1% BSA, 0,02% NaN₃) and 1.0 ml aliquots weredispensed to 12×75 mm polypropylene assay tubes. Separate dilutions ofhGH were made over a range from 120 mM to 0.002 mM. Aliquots (10 ml) ofhGH dilution or buffer only were then added to S237C-AF tubes and themixture incubated to equilibruim for 5 hours at 25° C. in the dark.After incubation, fluorescence measurements were made using anexcitation I of 490 nm and an emission I of 512 nm (bandwidths are 3 nmand 10 nm, respectively) using a Shimadzu RF5000USpectrofluorophotometer. F/F₀ values were calculated from triplicatereadings and plotted against hGH concentration. Preparation of S237C-AFwas as follows: Mutant S237C hGHbp was constructed and purified aspreviously described. A solution of 1 mg/ml S237C was brought to 25 mMcysteine HCl, 25 mM NaHCO₃ and incubated for 2 hours at 4° C. to deblockthe cysteine at position 237. The protein was de,salted using a PD10(Pharmacia) mini-column equilibrated with 50 mM tris.HCl pH 8 andimmeadiately reacted with 500 mM 5-IAF (Molecular Probes) for 16 hoursat 4° C. in dark. DTNB analysis of deblocked S237C prior to 5-IAFaddition showed an average of one free thiol group per S237C molecule(22 μM free SH vs. 17 μM S237C). The 5-IAF reacted S237C was purifiedfrom free fluorophore using another PD10 mini-column equilibrated with20 mM tris.HCl pH 7.5. Aliquots of purified S237C-AF were stored at -80°C. and thawed just prior to use. Adsorbtion spectrum analysis of theS237C-AF shows 0.84 mM fluorescein bound per 1.0 mM S237C using molarextinction coefficients of 71,300 (at 494 nm) and 64,800 (at 280 nm) andcorrecting for interfering 5-IAF adsorbance at 280 nM.

Here, hGH was serially diluted against a fixed 10 nM concentration ofS237C-AF and fluorescence quenching measured at equilibrium.Homoquenching increased with hGH addition and becames maximal (ΔF/F₀=11%) at 0.5 molar equivalents of hGH (5 nM). However, quite strikingly,this homoquenching is reversed at higher concentrations of hGHindicating hGH.(hGHbp)₂ dissociates to hGH.hGHbp monomeric complex inthe presence of excess hGH (i.e. hGH/hGHbp>0.5). The measuredfluorescence homoquenching reflect genuine Forster energy transfer asshown from experiments using a nonidentical donor/acceptor pair tomeasure both donor (S237C-AEDANS, ref.) fluorescence quenching andacceptor (S237C-AF) fluorescence enhancement. The increase in measuredfluoresence to values of F/F₀ >1, that occurs in the presence of a largeexcess of hGH (>70 nM), appears to be due to higher non-specific bindingof free S237C-AF versus bound hGH.S237C-AF complex. While thisphenomenon may slightly distort IC₅₀ values obtained in our assay (FIG.9), relative IC₅₀ values, which are the basis of our analysis, shouldremain uneffected.

In FIG. 9, the IC₅₀ determination for hGH induced dimerization ofS237C-AF was determined. Serial dilutions (3 fold) of S237C-AF (preparedas described for FIG. 8) in binding buffer (20 mM tris.HCl pH 7.5, 0.1%BSA, 0.02% NaN₃) were made over a range from 60 nM to 0.08 nM and 1.0 mlaliquots were dispensed to assay tubes. Simularly, hGH was seriallydiluted, but over a range from 3 μM to 0.004 μM. Aliquots (10 μl)of hGHdilution (giving 1:2 molar ratio hGH to S237C-AF) and buffer only wereadded to the S237C-AF containing assay tubes (in triplicate), mixed andincubated to equilibrium for 5 hours at 25° C. in dark. Afterequilibration, fluorescence was measured as previously described (FIG.8) except excitation bandwidth was 10 nM. IC₅₀ values are calculated asthe concentration of hGH giving half-maximal ΔF/F₀ values.

In Table 2, the IC₅₀ values for S237C-AF dimerization induced by varioushomolog substitution and deletion mutants of hGH are shown. Identitiesof hGH mutants are given as Δ, deletions and hPL, substitutions withhuman placental lactogen and hPRL, substitutions with human prolactin.Regions deleted or substituted are designated within parenthesis. IC₅₀values are determined as described for FIG. 2. Standard deviations aregenerally less than +/-50% of the reported value.

                  TABLE 2                                                         ______________________________________                                        Receptor dimerization determinants homolog-scan                                                          IC.sub.50 mutant                                   Mutant        Dimerization IC.sub.50                                                                     IC.sub.50 wt                                       ______________________________________                                        wt hGH        0.54         --                                                 Δ(1-8)hGH            >100                                               hPRL(12-19)   10           19                                                 hPRL(22-33)   .66          1.2                                                Δ(32-46)                                                                              .42          0.8                                                hPL(46-52)    .94          1.7                                                hPRL(54-74)   2.5          4.7                                                hPRL(88-95)   .72          1.3                                                hPRL(97-104)  1.6          2.9                                                hPL(109-112)  3.0          5.5                                                hPRL(111-129)              >100                                               hPRL(126-136) 1.2          2.2                                                hPRL(137-145) .69          1.3                                                hPRL(146-152) .51          0.9                                                ______________________________________                                    

Initially, a series of homolog-scan hGH mutants, with hPL and hPRLsegment substitutions, were tested in the S237C-AF based assay (table2). Three of these, hPRL(12-19), hPRL(54-74) and hPRL(111-129) causedsignificant reductions (18, 6 and >100 fold, respectively) in hormoneinduced hGHbp dimerization. However, hPRL 12-19 and hPRL54-74 disruptresidues crucial for primary site binding and have been shown tosubstantially reduce hGHbp affinity for this site. Losses in primarysite binding for these mutants appear to largely account for theobserved reductions in hGHbp dimerization. Indeed, the 500 pM IC₅₀observed for S237C-AF homoquenching by wild-type hGH is nearly identicalto that reported for primary site hGHbp affinity (Kd=400 pM).Furthermore, mutations of primary site determinants (e.g. R64A andK172A/F176A) which reduce binding affinity have also been shown toreduce dimerization, and an hGH mutant (E174A) shown to enhance hGHbpaffinity for the primary site also enhances dimerization as measured inour assay. The other homolog mutant, hPRL(111-129), although uneffectedfor primary site binding, shows evidence of heterogeneity when analyzedby size exclusion chromatography, with 90% forming only hGH.hGHbpcomplex, but the remaining 10% forming hGH.(hGHbp)₂. The existence of asub-fraction of this mutant with relatively intact secondary sitebinding suggest this mutant's effects may be attributable to proteinmisfolding or post translational modification.

In addition to the homolog-scan mutants, two hGH deletion analogs, oneremoving 8 residues from the N-terminus [Δ(1-8)] and the other, anatural variant (20K hGH, U.S. Pat. No. 4,446,235) deleting residues32-46, were tested (table 2). The Δ(1-8) mutant showed a dramaticreduction (>100 fold) in ability to induce hGHbp dimerization. Sincethis mutant has only a small effect on primary site binding (Kd_(mut)/Kd_(wt) =4), the loss in hGHbp dimerization appeared to be due todisruptions in secondary site hGHbp binding.

EXAMPLE 5 ALANINE SCANNING OF hGH VARIANTS

To elucidate specific side chains involved in secondary site hGHbpbinding we probed the domains identified in the Δ(1-8), hPRL(11-19) andhPRL(111-129) mutants by alanine scanning. Since the two domainsidentified by the homolog substitutions are helical, based on the X-raycrystal structure of porcine growth hormone and are highly amphipathic,we focused the mutants screened on those located at the hydrophilicsurface of these helices, where the residues are likely to be solventexposed. In addition to these domains we also screened 3 mutants nearthe C-terminus (E186A, S188A and F191A) since we lacked an appropriatehomolog substitution analog in this region.

From a set of 26 alanine mutants (table 3) we found two mutants, F1A andI4A which cause large disruptions in hGHbp dimerization (33 and 56 fold,respectively and four others causing ≧2-fold reductions (L6A, R8A,D116A, E119A). The alanine scan shows that residues adjacent to F1A andI4A in the N-terminal domain, as well as residues in the C-terminaldomains of helices A and C, do not contribute significantly to secondarysite hGHbp binding. Additional data from an hGH analog [Δ(1,2], deletingF1 and P2 from the N-terminal domain show Δ(1,2) does not disruptdimerization any further than does F1A alone, indicating that thephenyalanine side chain is important, not the N-terminal amine orcarbonyl group. The alanine scan analysis reveals that the hGHdeterminants most responsible for secondary site hGHbp binding andreceptor dimerization are the hydrophobic side chains at F1 and I4.These determinants are strikingly different from those crucial forprimary site binding, which consist of many residues (Matthews, B. W. J.Mol. Biol. 33:491-497 (1968)) of predominantly hydrophilic character(Boutin J. M. et. al., Cell 69:(1988)).

In Table 3, the IC₅₀ values for S237C-AF dimerization induced by alaninesubstituted hGH mutants are shown. Mutants are named by the wild-typeresidue and position in the amino acid sequence, followed by the mutantresidue (alanine in this case). Amino acids are designated by singleletter code as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe: G, Gly;H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg;S, Ser; T, Thr; V, Val; W, Trp and Y, Tyr. A mutant not expressed isdesignated NE. IC₅₀ numbers are calculated as described in FIG. 2.Standard deviations are generally less than +/-50% of the reported valueor as stated.

                  TABLE 3                                                         ______________________________________                                        Receptor dimerization functional determinants                                 Alanine scanning mutagenesis                                                                       IC.sub.50 mutant                                         Hormone Dimerization IC.sub.50                                                                     IC.sub.50 wt                                             ______________________________________                                        wt hGH  0.54         --                                                       F1A     7.5          14                                                       P2A     .58          1.1                                                      T3A     .72          1.3                                                      I4A     30           55                                                       P5A     .92          1.7                                                      L6A     1.4          2.5                                                      S7A     .37          0.7                                                      R8A     1.8          3.4                                                      F10A    .77          1.4         D11A                                         NE                                                                            N12A    .59          1.1                                                      L15A    .36          0.7                                                      R16A    .63          1.2                                                      H18A    .55          1.0                                                      R19A    .92          1.7                                                      H21A    .51          1.0                                                      D107A   .38          0.7                                                      N109A   .35          0.7                                                      Y111A   1.0          1.9                                                      D112A   .53          1.0                                                      K115A   .84          1.6                                                      D116A   3.1          5.7         E118A .96                                    1.8                                                                           E119A   1.1          2.0                                                      Q122A   .4           0.7                                                      T123A   .65          1.2                                                      R127A   .80          1.5                                                      E129A   .70          1.3                                                      D130A   .42          0.8                                                      E186A   .58          1.1                                                      S188A   .49          .9                                                       F191A                                                                         ______________________________________                                    

EXAMPLE 6 HOMOQUENCHING OF FLUORESENCE

Sequential hGH additions are made to a fixed concentration of S237C-AF(100 nM), and fluorescence homoquenching monitored in real time (Example4), show rapid equilibration times (<3 minutes) for hGH induceddimerization and slow equilibration times (>30 minutes) for subsequentreversal of dimerization by excess hGH (i.e. hGH/hGHbp>0.5) Thissuggests that reversal of dimerization is off-rate limited according tothe mechanism

    HR.sub.1 R.sub.2 →HR.sub.X +R→.sup.+excess H →2HR

where stoichiometric binding competes with dimerization under conditionsof excess hGH (H=hGH, R=free hGHbp, R₁ =primary site hGHbp, R₂=secondary site hGHbp). We know primary site stoichiometric bindingoccurs and therefore should compete with hGH(hGHbp)₂ complex formation.To determine if stoichiometric secondary site binding can occur wetested an analog, engineered to remove the primary site, for ability tocompete for dimerization. K172A/F176A, a double mutant with mutations inthe middle of the primary site which reduce hGHbp affinity 500 fold,retains the secondary site. FIG. 10 shows that hGHbp dimerization cannot be reversed by excess K172NF176A even when present at a 160 foldexcess (800 nM). By contrast a known hPL variant, containing anengineered primary site but lacking secondary site determinantsefficently blocks dimerization with an IC₅₀ of 20 nM (4 fold excess).This data demonstrates that stoichiometric secondary site binding doesnot occur and that dimerization must proceed by the sequential bindingmechanism:

    H+R→HR.sub.1 →.sup.+R →HR.sub.1 R.sub.2

Since secondary site hGHbp binding requires stoichiometric primary sitecomplex formation this binding event must be dependent upon determinantspresent in hGH.(hGHbp) and not hGH alone. As such these determinantsmust be introduced from primary site hGHbp and/or a conformationalchange elicited by the first hGHbp binding event.

EXAMPLE 7 hGH-hGHbp AMINO ACID INTERACTION BASED ON X-RAYCRYSTALLOGRAPHY

The formation of hGH(hGHbp)₂ crystals permits the determination of thethree-dimensional structure of the hGH(hGHbp)₂ complex using x-raychrstailographic techniques following the methods described in Blundelland Johnson, Academic Press, London, 1976. Crystals of the complex weregrown using a combination of vapor diffusion in sitting drops along withrepeat seeding. The crystal stock solution was prepared by adding hGHbpto the met-hGH in a slight 2:1 molar excess and allowed to incubate at4° C. for 24 hrs. The complex was then concentrated and loaded onto asize exclusion column (G75-120 Sephadex (Sigma)) that was equilibratedwith 120 mm NaCl, 20 mm sodium acetate pH 5.5, 1 mM PMSF. The fractionscontaining the complex were then pooled, concentrated and desalted onto50 mM sodium acetate pH 5.5, 1 mM PMSF. The concentration of the complexin the resulting stock solution was 4 mg/ml (E₂₈₀ (0.1%)=1.67 cm⁻¹). Thestock solution of complex was diluted to 1.7 mg/ml using 0.1M Bis-trispH 6.5, to which saturated ammonium sulfate (ultrapure (Schwarz-Mann))was added to make a 10% saturated solution. MPD (Aldrich) was added to afinal concentration of 1%. Fifty microliters of the mixture were thenpipetted into a Pyrex glass spot plate and allowed to equilibrateagainst 40% saturated ammonium sulfate for 2 days at room temperature ina 150 mm×25 mm plastic culture dish before seed crystals wereintroduced. Within two weeks, crystals were obtained with dimensions of1 mm×0.5 mm×0.1 mm, that diffract to 2.7 Å on a rotating anode generatoroperated at 45 kV, 110 mA.

The three dimensional polypeptide structure of the hGH(hGHbp)₂ crystalstructure is illustrated in FIG. 11. The central top region, in thickerlines, represents the hGH molecule; the alpha helices are clearlyvisible. This hGH molecule is bound to two hGHbp molecules: one at theleft hand side, and one at the right. Each of these hGHbp molecules hastwo domains linked by a single strand; the top domains are at the sameheight as the hGH molecule, the other domains are oriented verticallyand stick out towards the bottom of the figure; These last two domainsof the hGHbp contact each other at the very bottom of FIG. 11. Thiscontact at the bottom constitutes the only contact region between thetwo hGHbp molecules and the points of contact are discussed below. Basedupon this structure an analysis of the interacting amino acids of thethree polypeptides was made. They fall into three categories: 1)interactions between hGH and hGHbp1(the first hGHbp to bind)listed inTable 4; 2) interactions between hGH and hGHbp₂ (second hGHbp to bind)listed in Table 5; and 3) interactions between the two hGHbp in thecomplex listed in Table 6. Tables 4 and 5 disclose the unique individualhGH amino acids binding to the indicated unique hGHbp amino acids. Theparticular moiety bound and the nature of the chemical interaction arealso listed. The nomenclature follows standard amino acid single letternomenclature and the number of the amino acid when numbered from theamino terminus of the natural hGH or hGHpb. The remaining terms inTables 4, 5 and 6 are defined as follows: MC=main chain, SC=side chain,SS=disulfide, HB=hydrogen bond, SB=salt bridge, VW=van der Waals. Thesetables are not exclusive of all site 1 and 2-affecting residues.

                  TABLE 4                                                         ______________________________________                                        SITE 1 Interactions                                                           hGH      moiety  hGHbp1      moiety                                                                              interaction                                ______________________________________                                        H18      SC      R217        SC    VW                                                  SC      N218        SC    VW                                         H21      SC      N218        MC    VW                                                  SC      N218        SC    VW                                         Q22      MC      N218        SC    VW                                         F25      SC      S119        MC    VW                                                  SC      G120        MC    VW                                         K41      SC      E127        SC    SB                                         Y42      SC      K121        MC    VW                                                  SC      K121        SC    VW                                                  SC      C122        MC    VW                                                  SC      C122        SC    VW                                         L45      SC      P106        SC    VW                                                  SC      C122        MC    VW                                         Q46      SC      E120        SC    HB                                                  SC      C108-C122   SS    VW                                         P61      SC      S102        MC    VW                                                  SC      S102        SC    VW                                         S62      SC      R43         MC    HB                                                  SC      E244        SC    VW                                                  SC      W169        MC    VW                                         N63      MC      W169        SC    VW                                                  SC      W169        SC    VW                                                  SC      E244        SC    VW                                         E66      SC      W169        SC    VW                                         R167     SC      E127        SC    SB                                         K168     SC      W104        MC    HB,VW                                               SC      W104        SC    VW                                         D171     SC      R43         SC    SB                                                  SC      W104        SC    VW                                         K172     SC      W104        MC    VW                                                  SC      W104        SC    VW                                         T175     SC      R43         SC    HB                                                  SC      W104        SC    VW                                                  SC      W169        SC    VW                                         R178     SC      I165        MC    HB                                                  SC      G168        MC    VW                                         C182-C189                                                                              SS      K167        MC    VW                                         ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        SITE 2 Interactions                                                           hGH     moiety  hGHbp2      moiety                                                                              interaction                                 ______________________________________                                        T 3     SC      P106        SC    VW                                          I4      SC      F123        MC    VW                                          L6      SC      S124        SC    VW                                          L9      SC      W104        SC    VW                                          N12     SC      R143        SC    HB                                                  SC      W169        SC    VW                                                  MC      W169        SC    VW                                          L15     SC      W169        SC    VW                                          R16     SC      W169        SC    VW                                                  SC      E44         SC    SB                                          R19     SC      N166        SC    HB                                                  SC      K167        MC    VW                                                  SC      K167        SC    VW                                          Q22     SC      Q166        SC    VW                                          Y103    SC      Y164        MC    VW                                                  SC      I165        SC    VW                                                  SC      N166        MC    VW                                          N109    SC      K167        SC    HB                                          D116    SC      W104        SC    VW                                          D119    SC      W104        SC    VW                                                  SC      S102        SC    HB                                          G120    MC      W104        SC    VW                                          T123    SC      W104        SC    VW                                          ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Binding Protein Interactions                                                  hGHbp1   moiety  hGHbp2      moiety                                                                              interaction                                ______________________________________                                        S145     SC      D152        SC    VW                                                  SC      Y200        SC    VW                                         L146     SC      H150        SC    VW                                                  MC      S201        SC    HB                                         T147     SC      H150        SC    VW                                                  SC      D152        SC    HB                                         H150     SC      L142        SC    VW                                                  SC      N143        SC    HB                                                  SC      D152        SC    VW                                                  SC      Y200        SC    VW                                         D152     SC      Y200        SC    HB                                         Y200     SC      L192        SC    VW                                                  SC      V197        SC    VW                                                  SC      P198        SC    VW                                         S201     MC      P198        SC    VW                                                  MC      Y200        SC    HB?                                                 MC      Y200        SC    VW                                         ______________________________________                                    

EXAMPLE 8 USE OF MONOCLONAL ANTIBODY TO STIMULATE hGH RECEPTOR

The assay of the present invention may be used to screen monoclonalantibodies that are directed against growth hormone receptors. Theresulting monoclonal antibodies can then be evaluated in vivo forrelative ability to promote growth. The monoclonal antibody MAb 263(Agen Biochemical Ltd, Queensland, Australia) was made using as animmunogen the glycosylated rat and rabbit receptor. When MAb263 wasadministered daily by s.c. injection to hypophysectomized rats, at adosage equivalent on a molar basis to an hGH dose of 155 μg/Kg in rat,there was a significant body weight gain as shown in FIG. 12.

Two groups of eight rats each were given excipient buffer (10 mM Tris,pH 8, 0.1% bovine serum albumin) either with or without MAb 263 (1.05mg/kg). The rats were given food and water on demand. Daily weight isshown in FIG. 12. At the end of day six the rats were weighed with theresults in Table 7 below.

                  TABLE 7                                                         ______________________________________                                        Monoclonal Antibody Induced Weight Gain                                       Group   Body Weight Gain/Rat                                                                          Percent Weight Gain                                   ______________________________________                                        MAb263   6.075 g +/- 1.97 g                                                                           6.47 +/- 1.97%                                        Control 0.9875 g +/- 2.16 g                                                                           1.01 +/- 2.30%                                        ______________________________________                                    

Therefore, monoclonal antibody directed against the growth hormonereceptor can be administered to produce weight gain.

EXAMPLE 9 CELLULAR ASSAY FOR AGONIST OR ANTAGONIST ACTIVITY

A novel cell-based bioactivity assay system is provided herein. It isbased on a hybrid receptor (U.S. Pat. No. 4,859,609) transformed cellline that comprises an extracellular, GH-binding domain of the GHreceptor fused at its C-terminus to a hormone or cytokine receptor,e.g., that of EPO, alpha interferon, beta interferon, GM-CSF, C-CSF,prolactin, placental lactogen or interleukins 2, 3, 4, 6 or 7, the cellline ordinarily being responsive to the hormone or cytokine andordinarily containing the receptor for the hormone or cytokine. Usually,only the transmembrane and endoplasmic portions of the hormone orcytokine receptor are used, fused at their N-terminus to the GH receptorfragment. The responsive feature of the cell is any measurablecharacteristic, e.g., changes in membrane characteristics,proliferation, mitotic features, release of analytes (e.g.,degranulation) and the like.

The hGH receptor belongs to a large family of receptors of hematopoieticorigin (7), that includes the interleukin-3 (IL-3) and granulocytecolony stimulating factor (G-CSF) receptors. Nagata and coworkers (8)showed that an IL-3 dependent myeloid leukemia cell-line (FDC-P1)transfected with the full-length murine G-CSF receptor proliferates byaddition of G-CSF without IL-3. A hybrid receptor (U.S. Pat. No.4,859,809 and 5,030,576) was constructed by Drs. Etsuko Ishizaka-Ikedaand Shigekazu Nagata of the Osaka Bioscience Institute containing thehGHbp linked to a form of the mG-CSF receptor missing the G-CSF bindingdomain but containing the three extracellular fibronectin repeats, thetransmembrane and intracellular domains. The fibronectin domains are notinvolved in binding of G-CSF but are required for good expression of themG-CSF receptor (8).

The hybrid receptor was constructed from cDNA containing exons 1 through5 of the hGH receptor (that encodes the secretory signal and theextracellular hGH binding domains) linked to exons 7 through 15 of themG-CSF receptor (that encodes the three fibronectin domains plus theentire transmembrane and intracellular domains). Sequences derived fromthe hGH receptor (Leung D., et al., Nature 330:537 (1987)) were clonedby PCR into the vector, pBOS-I62 (8), which allowed expression of thehybrid receptor in FDC-P1 cells. A single cysteine was produced at thejunction of the two receptor fragments. Transfection and culturing ofstable FDC-P1 cell-lines were as described (infra).

Competitive displacement of [¹²⁵ I]hGH from hybrid-receptors on wholecells was used to establish the affinity and the approximate number ofreceptors per cell. Cells grown with IL-3 were washed before assay withphosphate buffered saline (PBS) plus 10% FBS. Cells were incubated(1.2×10⁶ /ml) with serial dilutions of hGH in the presence of 20 pM [¹²⁵I]hGH (Y103A) for 18 h at 4° C. Cells were then washed with PBS twice toremove the excess label. Y103A was used to prevent iodination of Y103which would partially block the binding of the second hGHbp (9).

In several independent binding experiments the apparent K_(d) value forhGH was 0.1±0.03 nM and there were 1000±300 receptors per cell. Thisaffinity is about 3-fold stronger than hGH binding to the soluble hGHbpand may reflect an avidity effect for binding of hGH to receptors oncells. Non-transfected cells lacked specific binding sites for hGH (9).FIG. 13 shows the effect of increasing hGH concentrations on the abilityof hGH to induce cell proliferation. At low concentrations, hGH acts asa potent agonist in this assay with an EC₅₀ of ˜20 pM, a value somewhatlower than the apparent K_(d) on whole cells (˜100 pM). This couldreflect that maximal cell-proliferation may occur at less than 100%receptor occupancy.

Mutational analysis (3, 4) and structural studies (5) show that each hGHmolecule is bivalent in that it contains two separate sites for bindingthe hGHbp. In contrast, the hGHbp is effectively univalent because eachuses virtually the same determinants to bind either Site 1 or Site 2 onhGH. Excess hGH will dissociate the hGH.(hGHbp)₂ complex to form ahGH.hGHbp complex in which hGH is bound exclusively via Site 1 to thehGHbp. Thus, we predicted that excess hGH should antagonize signaling(FIG. 1). Indeed, at very high hGH concentrations the proliferationactivity is lost (IC₅₀ @2 μM). IL-3 induced cell proliferation is notaltered in the presence of high concentrations of hGH (8 μM) indicatinghGH is not toxic to cell proliferation. Neither the agonist norantagonist inflection points depend on the cell density (FIG. 13)indicating the effect does not involve cross-linking receptors betweencells or other cell-cell interactions. Furthermore, FDC-P1 cellscontaining the full-length mG-CSF receptor do not respond to hGH, andneither do cells containing the hybrid receptor respond to G-CSF.

EXAMPLE 10

To further investigate the requirement for dimerization of the hGHbp forsignaling in the hybrid receptor cell proliferation assay we utilizedbivalent monoclonal antibodies (MAbs) and univalent fragments derivedfrom them (FAbs) that were directed against the hGHbp. Addition ofincreasing concentrations of three of four different anti-receptor MAbsat low concentrations were as potent as hGH in inducing cellproliferation (Table 8).

                  TABLE 8                                                         ______________________________________                                        Summary of dose response data for a variety of anti-hGH                       receptor MAbs, FAbs (16) and hGH mutants (17) to stimulate                    proliferation of FDC-P1 cells containing the hGH-mG-CSF                       hybrid receptor.                                                              None indicates no effect was observed and                                     ND indicates not determined.                                                                    Max. response                                                                           relative                                                                             Self-                                      Protein   K.sub.d (nM)*                                                                          EC.sub.50                                                                              to hGH antagonism                                 ______________________________________                                        MAb263    0.6      0.3 nM   (110)  >10 μM                                  MAb13E1   3.2      0.8 nM   (100)  >>10 μM                                 MAb3D9    2.2      0.8 nM   (80)   0.2 μM                                  MAb5      0.7      ˜1 nM                                                                            (10)   20 nM                                      FAb263    ND       >1.5 μM      ND                                         FAb13E1   ND       >3 μM        ND                                         FAb3D9    ND       >0.1 μM      ND                                         FAb5      ND       >1 μM        ND                                         hGH       0.3      20 pM           2 μM                                    K172A/F176A                                                                             200      25 nM           None                                       G120R     0.3      None            --                                         H21A/R64K/                                                                              0.01     20 pM           60 nM                                      E174A                                                                         H21A/R64K/                                                                              0.01     None            --                                         E174A/G120R                                                                   ______________________________________                                         *K.sub.d values for MAbs binding to the hGHbp were taken from ref. 13.        K.sub. d value for hGH and variants were measured using a [.sup.125 I]hGH     competitive displacement assay where hormone bound to hGHbp was               precipitated with MAb5 (4, 12). This gives the affinity for the monomeric     hGHhGHbp complex.                                                              Values for EC.sub.50 were taken from titration curves shown for example      in FIG. 13 (except for FAbs which are not shown) and represent the            halfmaximal concentration for stimulation of cell proliferation. Data are     the mean of triplicate tubes and the S.D. are within 15% of mean. Values      shown with ">" indicate that we could not go to high enough concentration     of protein to complete the titration curve. For these cases we only repor     limit estimates of the EC.sub.50.                                              Self-antagonism refers to the halfmaximal concentration leading to           inhibition of cellproliferation at high concentration.                   

MAb5 and 263 were from Agen, Inc. (New Jersey) and have been describedby Waters and coworkers (Barnard, R., et al., Endocrinology115:1805-1813 (1984); Barnard, R., et al., Biochem. J. 231:459-468(1985). MAbs 13E1 and 3D9 were from the Genentech hybridoma group andtheir properties have been described elsewhere (3). Briefly, MAbs werepurified from mouse ascites fluid by binding to Protein-A Sepharose andelution with 0.1M acetate (pH 3.0). FAb fragments were prepared bytreating MAbs with dithiothreitol-activated papain (50:1 wt MAb/wtpapain) in PBS plus 10 mM cysteine for 1 h. Digestions were stopped byadding 0.1M iodoacetamide. The Fc and residual MAb was removed byadsorption onto Protein-A Sepharose twice, followed by gel filtration onSuperose 12 (Pharmacia).

The EC₅₀ value for each MAb (0.3 to 1 nM) was usually somewhat less thanthe K_(d) as determined by ELISA (Table 8). As with hGH, this mayreflect avidity effects on whole cells, and/or that maximal signaling isachieved at less than 100% receptor occupancy. At much higherconcentrations (20 nM to >10 μM) two of these MAbs lost activitypresumably because excess MAb blocks receptor cross-linking due tomonovalent attachment to hGHbp. Corresponding monovalent FAb fragmentswere virtually inactive (Table 8) further indicating that bivalency isrequired for signaling activity.

The differences in dose response curves for these MAbs can be explainedby the different ways they bind to the hGHbp. MAb5 prevents binding of asecond hGHbp to the hGH.hGHbp complex (3), possibly by binding to theregion where both receptors contact each other (FIG. 11). The fact thatMAb5 is the least efficient may indicate the receptors need to closelyapproach each other for maximal signaling. MAb13E1 blocks hGH binding(11) and mimics the effect of hGH. This MAb showed a broad plateau andno antagonistic phase probably because we could not go to high enoughMAb concentrations to observe one. We suggest this neutralizing MAbbinds like hGH to form very stable receptor dimers. In contrast, MAbs263 and 3D9 bind away from the hormone-receptor interfaces and showsimilar agonistic and antagonistic phases. These two phases are not aswidely separated as for hGH perhaps because the dimers do not have theoptimal receptor-receptor contacts. The tact that MAbs 263 and 3D9 areagonists suggest that the structural requirements to form active dimersare rather loose.

FAb fragments derived from MAb13E1 or MAb5 antagonize hGH-induced cellproliferation whereas those derived from MAbs 263 and 3D9 do not (Table9). These studies are consistent with the fact that the epitopes for MAb13E1 and MAb 5 block hormone-receptor or receptor-receptor interlaces.

                  TABLE 9                                                         ______________________________________                                        Summary of antagonist effects of FAbs and hGH analogs that                    block hGH-induced cell proliferation of FDC-P1 cells                          containing the hybrid hGH-mG-CSF receptor. Cells were                         incubated with 1 nM hGH plus increasing concentrations of                     FAb or hGH analog The half-maximal inhibitory concentration                   is that required to block 50% of the cell-proliferation activity              of hGH. None indicates no inhibition was observed for up to                   10 μM FAb or hGH analog.                                                   Protein                  IC.sub.50                                            ______________________________________                                        FAb263                   None                                                 FAb13E1                  0.8 μM                                            FAb5                     0.2 μM                                            FAb3D9                   None                                                 hGH                      2 μM                                              K172A/F176A              None                                                 G120R                    20 nM                                                H21A/R64K/E174A          60 nM                                                H21A/R64K/E174A/G120R    2 nM                                                 ______________________________________                                    

EXAMPLE 11

To further determine the structural requirements on hGH for dimerization(FIG. 11) we examined mutants of hGH that were designed to reducebinding of receptors to Site 1 or Site 2. The double mutant(K172A/F176A), which preserves Site 2 determinants but alters importantside-chains in Site 1 (12), promotes cell proliferation but the EC₅₀ isshifted to a concentration about 10³ -fold higher than wild-type hGH(Table 8). This is consistent with the 560-fold reduction in the K_(d)for Site 1 binding as measured in vitro (12). We could not go to highenough concentrations to observe an inactive phase in the titration withK172A/F176A. The single hGH mutant (G120R) retains a functional Site 1but sterically blocks Site 2. This mutant is virtually inactive at anyconcentration. Thus, binding to either Site 1 or Site 2 is necessary butnot sufficient for promoting cell proliferation.

The sequential signaling mechanism predicts that mutants blocked in Site2 binding (but not Site 1 binding) should antagonize hGH-induced cellproliferation. To test this we cultured cells with enough hGH (1 nM) tosupport 90% of maximal cell proliferation plus increasing concentrationsof wild-type hGH or the mutants in Site 1 (K172A/F176A) or Site 2(G120R). As we expected the Site 2 mutant antagonizes hGH whereas theSite 1 mutant is totally ineffective. In fact, the Site 2 mutant isnearly 100-fold more potent as an antagonist than wild-type hGH (IC₅₀ is20 nM for G120R versus 2 μM for hGH; Table 9). This was not unexpectedto us because once G120R is bound it can not dimerize and agonize thereceptor. Thus, competition between G120R and hGH is more confined tofree hormone molecules binding through Site 1. In contrast, for hGH tobe antagonistic free hormone needs to react with unoccupied receptorsbefore bound hGH intermediate does. This requires high concentrations ofhGH.

Although G120R is a much better antagonist than hGH, the concentrationof mutant required for 50% antagonism was about 20-times higher thanthat of hGH in the assay (Table 9). This may reflect the fact that hGHis bound more tightly in the dimeric hGH.(receptor)₂ complex than G120Ris in the monomeric G120R.receptor complex. Alternatively, maximalsignaling may not require 100% receptor occupancy. In either caseimproving the affinity for Site 1 in the G120R mutant will make it amore potent antagonist.

hGH variants have been produced by mutagenesis (4, 13) that bind moretightly to the hGHbp via Site 1. A combination of these variants(H21A/R64K/E174A) binds 30-times more tightly to the hGHbp (Table 9).This variant had an EC₅₀ comparable to hGH but an IC₅₀ forself-antagonism that is about 30 times lower than hGH. This isconsistent with the notion that self-antagonism results from competitionbetween Site 2 on bound hormone-receptor intermediate and free Site 1 onthe soluble hormone. The fact that improving Site 1 binding did notimprove this hormone as an agonist could reflect that receptordimerization is rate limiting and that it therefore is desirable tointroduce agonist mutations into both Sites 1 and 2. We further mutatedthis variant to contain G120R. The tetra-mutant variant was 10-fold morepotent than G120R as an hGH antagonist (FIG. 14, Table 9). This isfurther evidence for the importance of Site 1 binding affinity forantagonism.

Our studies indicate that the antagonism or self-antagonism caused byhGH, MAbs and their derivatives is the result of blocking receptordimerization and not receptor-down regulation. Firstly, cells propagatedwith IL-3 instead of hGH do not show a greater hGH response or hGHreceptor number. Receptor down-regulation is usually tightly coupled toreceptor activation. In this case one may expect the antagonisticportion of the dose response curve for hGH to start at physiologicallyrelevant concentrations of hGH (not 1 μM). Moreover, the ratio of EC₅₀to IC₅₀ for each of the MAbs and hGH varies widely showing that receptoractivation can be readily uncoupled from inhibition by simply alteringbinding properties. Finally, the G120R mutant is inactive yet it is amore potent antagonist than hGH, and pretreatment of cells with G120Rdoes not enhance its antagonistic effect. Thus, the antagonistic effectof G120R is not consistent with simple receptor down-regulation. It ispossible that other ligands that exhibit self-antagonism at highconcentrations may involve blocking of receptor dimerization, and thisserves as an additional basis for identifying ligands that are useful inthe practice of this invention.

CITATIONS

1. Melmed, S., New Engl. J. Med. 322:966-977 (1990); Frohman, L. A., J.Clin. Endo. Metab. 1175-1181 (1991).

2. Wells, J. A., et al., Recent Prog. Hormone Res. 52, in press (1992).

3. Cunningham, B. C., et al., Science 254:821-825 (1992).

4. Cunningham B. C., et al., Science 244:1081-1085 (1989).

5. de Vos, A. M., et al., Science 255:306-312 (1992).

6. Yarden Y., et al., Ann. Rev. Biochem. 57:443 (1988); Ullrich A., etal., Cell 61:203 (1990).

7. Bazan, J. F., Proc. Natl. Acad. Sci. U.S.A. 87:6934 (1990); Cosman D.et al. , Trends Biochem. Sci. 15:265 (1990); Patthy, L. Cell 61:13(1990).

8. Fukunaga, R., et al., EMBO J. 10:2855-2865 (1991).

9. Bass, S. H., et al., Proc. Natl. Acad. Sci. U.S.A. 88:4498-4502(1991).

10. Cunningham B. C., et al., Proc. Natl. Acad. Sci. U.S.A. 88:3407-3411(1991).

11. Cunningham, B. C., et al., Science 247:1461-1465 (1990).

12. Elberg, et al., JBC. 265:14770 (1990)

13. N. Itoh, et al., Science 247:324 (1990)

What is claimed is:
 1. A method for determining whether a candidate ligand that binds to a first receptor polypeptide antagonizes a native, monomeric ligand having four amphipathic alpha helices, wherein said native ligand forms ternary complexes with said first receptor polypeptide and a second receptor polypeptide, said method comprising:(a) contacting said candidate ligand with said first and second receptor polypeptides or receptor polypeptide variants to produce candidate ligand:receptor complexes, said receptor polypeptide variants having their transmembrane domain deleted or otherwise rendered incapable of membrane insertion or hydrophobic association; and (b) determining whether said candidate ligand:receptor complexes comprise one or two receptor polypeptides, the presence of only one receptor polypeptide indicating antagonist activity.
 2. The method of claim 1 wherein the number of receptor polypeptides in said candidate ligand:receptor complexes is detected by a method selected from the group consisting of fluorescence energy transfer, calorimetry, sedimentation equilibrium, gel filtration, and electrophoresis.
 3. The method of claim 1 wherein the candidate ligand is an amino acid sequence variant of the native ligand.
 4. The method of claim 1 wherein the native ligand is selected from the group consisting of growth hormone, prolactin, placental lactogen, EPO, alpha interferon, beta interferon, GM-CSF, G-CSF, and interleukins 2, 3, 4, 6 and
 7. 5. The method of claim 1 wherein the number of receptor polypeptides in said candidate ligand:receptor complexes is determined in a competition assay.
 6. The method of claim 1 wherein the candidate ligand is contacted with a receptor polypeptide variant corresponding to the extracellular domain of the receptor.
 7. The method of claim 1 wherein said first and second receptor polypeptides or receptor polypeptide variants are each labelled with a fluorescent label and the number of receptor polypeptides in said candidate ligand:receptor complexes is detected by assaying homoquenching of the label.
 8. A method for determining whether a candidate ligand that binds to a first receptor polypeptide potentially has agonist activity corresponding to a native, monomeric ligand having four amphipathic alpha helices, wherein said native ligand forms ternary complexes with a first receptor polypeptide and a second receptor polypeptide, said method comprising:(a) contacting said candidate ligand with a first and second receptor polypeptide or receptor polypeptide variants to produce candidate ligand:receptor complexes, said receptor polypeptide variants having their transmembrane domain deleted or otherwise rendered incapable of membrane insertion or hydrophobic association; and (b) determining whether said candidate ligand:receptor complexes comprise one or two receptor polypeptides, the presence of two receptor polypeptides indicating potential agonist activity.
 9. The method of claim 8 wherein the number of receptor polypeptides in said candidate ligand:receptor complexes is detected by a method selected from the group consisting of fluorescence energy transfer, calorimetry, sedimentation equilibrium, gel filtration, and electrophoresis.
 10. The method of claim 8 wherein the candidate ligand is an amino acid sequence variant of the native ligand.
 11. The method of claim 8 wherein the native ligand is selected from the group consisting of EPO, alpha interferon, beta interferon, GM-CSF, G-CSF, and interleukins 2, 3, 4, 6 and
 7. 12. The method of claim 8 wherein the candidate ligand is contacted with a receptor polypeptide variant corresponding to the extracellular domain of the receptor.
 13. The method of claim 8 wherein said first and second receptor polypeptides or receptor polypeptide variants are each labelled with a fluorescent label and the number of receptor polypeptides in said candidate ligand:receptor complexes is detected by assaying homoquenching of the label.
 14. A method for determining whether a candidate ligand that binds to a first receptor polypeptide has agonist activity corresponding to a native ligand selected from the group consisting of growth hormone, prolactin, and placental lactogen, wherein said native ligand forms ternary complexes with a first receptor polypeptide and a second receptor polypeptide, said method comprising:(a) contacting said candidate ligand with said first and second receptor polypeptides or receptor polypeptide variants to produce candidate ligand:receptor complexes, said receptor polypeptide variants having their transmembrane domain deleted or otherwise rendered incapable of membrane insertionor hydrophobic association; and (b) determining whether said candidate ligand:receptor complexes comprise one or two receptor polypeptides, the presence of two receptor polypeptides indicating agonist activity.
 15. The method of claim 14 wherein the number of receptor polypeptides in said candidate ligand:receptor complexes is detected by a method selected from the group consisting of fluorescence energy transfer, calorimetry, sedimentation equilibrium, gel filtration, and electrophoresis.
 16. The method of claim 14 wherein the candidate ligand is an amino acid sequence variant of the native ligand.
 17. The method of claim 14 wherein the candidate ligand is contacted with a receptor polypeptide variant corresponding to the extracellular domain of the receptor.
 18. The method of claim 14 wherein said first and second receptor polypeptides or receptor polypeptide variants are each labelled with a fluorescent label and the number of receptor polypeptides in said candidate ligand:receptor complexes is detected by assaying homoquenching of the label. 